Chordin-like molecules and uses thereof

ABSTRACT

The present invention provides Chordin-Like (CHL) polypeptides and nucleic acid molecules encoding the same. The invention also provides selective binding agents, vectors, host cells, and methods for producing CHL polypeptides. The invention further provides pharmaceutical compositions and methods for the diagnosis, treatment, amelioration, and/or prevention of diseases, disorders, and conditions associated with CHL polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/724,915, filed Nov. 28, 2000, now allowed, which claims the benefitunder 35 U.S.C. 119(e) of U.S. Provisional Application Ser. No.60/169,494, filed Dec. 7, 1999, all of which are incorporated in theirentirety by reference herein.

FIELD OF THE INVENTION

The present invention relates to Chordin-Like (CHL) polypeptides andnucleic acid molecules encoding the same. The invention also relates toselective binding agents, vectors, host cells, and methods for producingCHL polypeptides. The invention further relates to pharmaceuticalcompositions and methods for the diagnosis, treatment, amelioration,and/or prevention of diseases, disorders, and conditions associated withCHL polypeptides.

BACKGROUND OF THE INVENTION

Technical advances in the identification, cloning, expression, andmanipulation of nucleic acid molecules and the deciphering of the humangenome have greatly accelerated the discovery of novel therapeutics.Rapid nucleic acid sequencing techniques can now generate sequenceinformation at unprecedented rates and, coupled with computationalanalyses, allow the assembly of overlapping sequences into partial andentire genomes and the identification of polypeptide-encoding regions. Acomparison of a predicted amino acid sequence against a databasecompilation of known amino acid sequences allows one to determine theextent of homology to previously identified sequences and/or structurallandmarks. The cloning and expression of a polypeptide-encoding regionof a nucleic acid molecule provides a polypeptide product for structuraland functional analyses. The manipulation of nucleic acid molecules andencoded polypeptides may confer advantageous properties on a product foruse as a therapeutic.

In spite of the significant technical advances in genome research overthe past decade, the potential for the development of novel therapeuticsbased on the human genome is still largely unrealized. Many genesencoding potentially beneficial polypeptide therapeutics or thoseencoding polypeptides, which may act as “targets” for therapeuticmolecules, have still not been identified. Accordingly, it is an objectof the invention to identify novel polypeptides, and nucleic acidmolecules encoding the same, which have diagnostic or therapeuticbenefit.

Bone morphogenetic protein (BMP) is a member of the transforming growthfactor-beta family, which was originally identified as a factorpromoting bone formation from a cartridge implant (Wozney et al., 1988,Science 242:1528-34; Celeste et al., 1990, Proc. Nat. Acad. Sci. USA87:984347). BMP is also known to play an essential role during the earlyembryogenesis of the frog, the fly, and in mammals. The preciseconcentration of active BMP seems to be important for the specificationof particular cell types (Dale et al., 1992, Development 115:573-85;Dosch et al., 1997, Development 124:2325-34). An activity gradient ofBMP2/4 is observed in, for example, Xenopus embryos in which the lowestexpression is detected at the dorsal tip and the highest expression atthe ventral tip—establishing the dorsoventral axis determination in theembryo. In another example, the control of BMP concentration at specificsites of tissue development suggests a role for BMP in organogenesis.Control of BMP expression is achieved by either localized expression ofthe BMP gene products or through the influence of the BMP inhibitorchordin (CHD) (Sasai et al., 1994, Cell 79:779-90)—or short gastrulation(SOG) (Francois et al., 1994, Genes Dev. 8:2602-16).

CHD/SOG is a large secreted protein produced from the Spemann'sorganizer, the master-controlling region for the dorsoventral axisspecification at the gastrulation stage of Xenopus embryogenesis.CHD/SOG functions as a dorsalization factor, as does Noggin (Smith andHarland, 1992, Cell 70:829-40), which is also secreted from theorganizer. The Drosophila SOG has a transmembrane domain at itsamino-terminus, suggesting that it may be a type II transmembraneprotein (Francois et al., 1994, Genes Dev. 8:2602-16). It has beenproposed that the carboxyl-terminal side (extracellular domain) of theDrosophila SOG is cleaved off. However, Xenopus CHD (Sasai et al., 1994,Cell 79:779-90), Zebrafish CHD (Schulte-Merker et al., 1997, Nature387:862-63), and murine CHD (Pappano et al., 1998, Genomics 52:236-39)do not contain the transmembrane domain. Instead, these proteins have asignal peptide, and are secreted directly. The CHD/SOG polypeptidecontains four repeats of the cysteine-rich domain (CR1-4) that is alsofound in a variety of extracellular matrix proteins such as collagen andthrombospondin.

CHD/SOG is known to bind to one of the ventralizing factors, BMP4(Piccolo et al., 1996, Cell 86:589-98). BMP4 has been shown to beessential for embryonic development of posterior-ventral mesoderm inmice (Winnier et al., 1995, Genes Dev. 9:2105-16). The binding ofCHD/SOG to BMP4 inhibits BMP4 activity by preventing BMP4 from bindingto its receptor (Piccolo et al., 1996, Cell 86:589-98). In this respect,the functional relationship between CHD/SOG and BMP4 resembles thatbetween OPG and OPGL, although CHD/SOG is not structurally related tothe BMP receptors. The binding affinity of CHD/SOG to BMP4 is specificand tight (Kd=3×10⁻¹⁰ M (Piccolo et al., 1996, Cell 86:589-98), andseems to require proteolysis in order to effectuate the release of boundBMP4. This proteolysis is achieved by a specific metalloprotease—Tolloid(TLD) or BMP1—that cleaves CHD/SOG to liberate either, or both, thefirst (CR1) and last (CR4) CR motifs (Piccolo et al., 1997, Cell 91:407-16). Whether or not CHD/SOG has other functions or an independentfunction through its own receptor remains to be determined.

One of the most important roles of CHD/SOG is to establish a BMP4morphogen gradient (Jones and Smith, 1998, Dev. Biol. 194:12-17). BMP4itself only migrates a short distance and seems to act essentially onthe cell autonomously (Jones et al., 1996, Curr. Biol. 6:1468-75). Incontrast, the BMP4 inhibitors Noggin and CHD/SOG appear to exert along-range effect, thereby forming an activity gradient of BMP4.

BMPs also play important roles outside of early embryogenesis, forexample in the organogenesis of lung, gut, kidney, skin, heart andteeth, as well as in the later stages of embryogenesis (Hogan, 1996,Genes Dev. 10:1580-94). Some BMPs are expressed in a very localizedfashion while others are expressed widely in a tissue. The importance ofthe localized action of BMP for organogenesis has been supported bytransgenic mouse experiments using constructs by which BMP concentrationis artificially elevated throughout the target tissue. In the case oflung, BMP4 is expressed in the distal tips of epithelium in thedeveloping lung, and when overexpressed with the surfactant protein Cpromoter, the development of a small lung in which the structuralorganization (i.e., branching) has been severely disrupted is observed(Bellusci et al., 1996, Development 122:1693-702). Since the putativeBMP-activity gradient could also be disrupted by the transgeneexpression, BMPs expressed widely in the tissue could also play a rolein the determination of the structural organization of a tissue.

Noggin is another BMP2/4 inhibitor secreted from Spemann's organizer(Zimmerman et al., 1996, Cell 86:599-606). The biological role of Nogginand its mode of action are similar to CHD/SOG in Xenopus. Although themost notable function of Noggin is, like CHD/SOG, dorsalization, Nogginnull-mutant mice have shown a bone phenotype (hyperplasia ofchondrocytes) instead of an early embryonic phenotype (McMahon et al.,1998, Genes Dev. 12:1438-52; Brunet et al., 1998, Science 280:1455-57).This suggests that CHL or even CHD might have a non-dispensable functionin the later stage of embryogenesis.

SUMMARY OF THE INVENTION

The present invention relates to novel CHL nucleic acid molecules andencoded polypeptides.

The invention provides for an isolated nucleic acid molecule comprisinga nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence as set forth in any of SEQ ID NO: 1, SEQ IDNO: 4, SEQ ID NO: 7, or SEQ ID NO: 11;

(b) the nucleotide sequence of the DNA insert in any of ATCC DepositNos. PTA-961, PTA-962, PTA-963, or PTA-964;

(c) a nucleotide sequence encoding the polypeptide as set forth in anyof SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12;

(d) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of any of (a)-(c); and

(e) a nucleotide sequence complementary to any of (a)-(c).

The invention also provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide which is at least about70 percent identical to the polypeptide as set forth in any of SEQ IDNO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12, wherein the encodedpolypeptide has an activity of the polypeptide set forth in any of SEQID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12;

(b) a nucleotide sequence encoding an allelic variant or splice variantof the nucleotide sequence as set forth in any of SEQ ID NO: 1, SEQ IDNO: 4, SEQ ID NO: 7, or SEQ ID NO: 11, the nucleotide sequence of theDNA insert in any of ATCC Deposit Nos. PTA-961, PTA-962, PTA-963, orPTA-964, or (a);

(c) a region of the nucleotide sequence of any of SEQ ID NO: 1, SEQ IDNO: 4, SEQ ID NO: 7, or SEQ ID NO: 11, the DNA insert in any of ATCCDeposit Nos. PTA-961, PTA-962, PTA-963, or PTA-964, (a), or (b) encodinga polypeptide fragment of at least about 25 amino acid residues, whereinthe polypeptide fragment has an activity of the polypeptide set forth inany of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12, or isantigenic;

(d) a region of the nucleotide sequence of any of SEQ ID NO: 1, SEQ IDNO: 4, SEQ ID NO: 7, or SEQ ID NO: 11, the DNA insert in any of ATCCDeposit Nos. PTA-961, PTA-962, PTA-963, or PTA-964, or any of (a)-(c)comprising a fragment of at least about 16 nucleotides;

(e) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of any of (a)-(d); and

(f) a nucleotide sequence complementary to any of (a)-(d).

The invention further provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide as set forth in any ofSEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 with at leastone conservative amino acid substitution, wherein the encodedpolypeptide has an activity of the polypeptide set forth in any of SEQID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12;

(b) a nucleotide sequence encoding a polypeptide as set forth in any ofSEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 with at leastone amino acid insertion, wherein the encoded polypeptide has anactivity of the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO:5, SEQ ID NO: 8, or SEQ ID NO: 12;

(c) a nucleotide sequence encoding a polypeptide as set forth in any ofSEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 with at leastone amino acid deletion, wherein the encoded polypeptide has an activityof the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQID NO: 8, or SEQ ID NO: 12;

(d) a nucleotide sequence encoding a polypeptide as set forth in any ofSEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 which has aC- and/or N-terminal truncation, wherein the encoded polypeptide has anactivity of the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO:5, SEQ ID NO: 8, or SEQ ID NO: 12;

(e) a nucleotide sequence encoding a polypeptide as set forth in any ofSEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 with at leastone modification selected from the group consisting of amino acidsubstitutions, amino acid insertions, amino acid deletions, C-terminaltruncation, and N-terminal truncation, wherein the encoded polypeptidehas an activity of the polypeptide set forth in any of SEQ ID NO: 2, SEQID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12;

(f) a nucleotide sequence of any of (a)-(e) comprising a fragment of atleast about 16 nucleotides;

(g) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of any of (a)-(f); and

(h) a nucleotide sequence complementary to any of (a)-(e).

The present invention provides for an isolated polypeptide comprising anamino acid sequence selected from the group consisting of:

(a) the amino acid sequence as set forth in any of SEQ ID NO: 2, SEQ IDNO: 5, SEQ ID NO: 8, or SEQ ID NO: 12; and

(b) the amino acid sequence encoded by the DNA insert in any of ATCCDeposit Nos. PTA-961, PTA-962, PTA-963, or PTA-964.

The invention also provides for an isolated polypeptide comprising theamino acid sequence selected from the group consisting of:

(a) the amino acid sequence as set forth in any of SEQ ID NO: 3, SEQ IDNO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, or SEQ ID NO: 14optionally further comprising an amino-terminal methionine;

(b) an amino acid sequence for an ortholog of any of SEQ ID NO: 2, SEQID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12;

(c) an amino acid sequence which is at least about 70 percent identicalto the amino acid sequence of any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ IDNO: 8, or SEQ ID NO: 12, wherein the polypeptide has an activity of thepolypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:8, or SEQ ID NO: 12;

(d) a fragment of the amino acid sequence set forth in any of SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 comprising at leastabout 25 amino acid residues, wherein the fragment has an activity ofthe polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ IDNO: 8, or SEQ ID NO: 12, or is antigenic; and

(e) an amino acid sequence for an allelic variant or splice variant ofthe amino acid sequence as set forth in any of SEQ ID NO: 2, SEQ ID NO:5, SEQ ID NO: 8, or SEQ ID NO: 12, the amino acid sequence encoded bythe DNA insert in any of ATCC Deposit Nos. PTA-961, PTA-962, PTA-963, orPTA-964, or any of (a)-(c).

The invention further provides for an isolated polypeptide comprisingthe amino acid sequence selected from the group consisting of:

(a) the amino acid sequence as set forth in any of SEQ ID NO: 2, SEQ IDNO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 with at least one conservativeamino acid substitution, wherein the polypeptide has an activity of thepolypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:8, or SEQ ID NO: 12;

(b) the amino acid sequence as set forth in any of SEQ ID NO: 2, SEQ IDNO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 with at least one amino acidinsertion, wherein the polypeptide has an activity of the polypeptideset forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ IDNO: 12;

(c) the amino acid sequence as set forth in any of SEQ ID NO: 2, SEQ IDNO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 with at least one amino aciddeletion, wherein the polypeptide has an activity of the polypeptide setforth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO:12;

(d) the amino acid sequence as set forth in any of SEQ ID NO: 2, SEQ IDNO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 which has a C- and/or N-terminaltruncation, wherein the polypeptide has an activity of the polypeptideset forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ IDNO: 12; and

(e) the amino acid sequence as set forth in any of SEQ ID NO: 2, SEQ IDNO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 with at least one modificationselected from the group consisting of amino acid substitutions, aminoacid insertions, amino acid deletions, C-terminal truncation, andN-terminal truncation, wherein the polypeptide has an activity of thepolypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:8, or SEQ ID NO: 12.

Also provided are fusion polypeptides comprising CHL amino acidsequences.

The present invention also provides for an expression vector comprisingthe isolated nucleic acid molecules as set forth herein, recombinanthost cells comprising the recombinant nucleic acid molecules as setforth herein, and a method of producing a CHL polypeptide comprisingculturing the host cells and optionally isolating the polypeptide soproduced.

A transgenic non-human animal comprising a nucleic acid moleculeencoding a CHL polypeptide is also encompassed by the invention. The CHLnucleic acid molecules are introduced into the animal in a manner thatallows expression and increased levels of a CHL polypeptide, which mayinclude increased circulating levels. Alternatively, the CHL nucleicacid molecules are introduced into the animal in a manner that preventsexpression of endogenous CHL polypeptide (i.e., generates a transgenicanimal possessing a CHL polypeptide gene knockout). The transgenicnon-human animal is preferably a mammal, and more preferably a rodent,such as a rat or a mouse.

Also provided are derivatives of the CHL polypeptides of the presentinvention.

Additionally provided are selective binding agents such as antibodiesand peptides capable of specifically binding the CHL polypeptides of theinvention. Such antibodies and peptides may be agonistic orantagonistic.

Pharmaceutical compositions comprising the nucleotides, polypeptides, orselective binding agents of the invention and one or morepharmaceutically acceptable formulation agents are also encompassed bythe invention. The pharmaceutical compositions are used to providetherapeutically effective amounts of the nucleotides or polypeptides ofthe present invention. The invention is also directed to methods ofusing the polypeptides, nucleic acid molecules, and selective bindingagents.

The CHL polypeptides and nucleic acid molecules of the present inventionmay be used to treat, prevent, ameliorate, and/or detect diseases anddisorders, including those recited herein.

The present invention also provides a method of assaying test moleculesto identify a test molecule that binds to a CHL polypeptide. The methodcomprises contacting a CHL polypeptide with a test molecule to determinethe extent of binding of the test molecule to the polypeptide. Themethod further comprises determining whether such test molecules areagonists or antagonists of a CHL polypeptide. The present inventionfurther provides a method of testing the impact of molecules on theexpression of CHL polypeptide or on the activity of CHL polypeptide.

Methods of regulating expression and modulating (i.e., increasing ordecreasing) levels of a CHL polypeptide are also encompassed by theinvention. One method comprises administering to an animal a nucleicacid molecule encoding a CHL polypeptide. In another method, a nucleicacid molecule comprising elements that regulate or modulate theexpression of a CHL polypeptide may be administered. Examples of thesemethods include gene therapy, cell therapy, and anti-sense therapy asfurther described herein.

In another aspect of the present invention, the CHL polypeptides may beused for identifying receptors thereof (“CHL polypeptide receptors”).Various forms of “expression cloning” have been extensively used toclone receptors for protein ligands. See, e.g., Simonsen and Lodish,1994, Trends Pharmacol. Sci. 15:437-41 and Tartaglia et al., 1995, Cell83:1263-71. The isolation of a CHL polypeptide receptor is useful foridentifying or developing novel agonists and antagonists of the CHLpolypeptide signaling pathway. Such agonists and antagonists includesoluble CHL polypeptide receptors, anti-CHL polypeptidereceptor-selective binding agents (such as antibodies and derivativesthereof), small molecules, and antisense oligonucleotides, any of whichcan be used for treating one or more disease or disorder, includingthose disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C illustrate the nucleotide sequence of the murine CHL gene(SEQ ID NO: 1) and the deduced amino acid sequence of murine CHLpolypeptide (SEQ ID NO: 2). The predicted signal peptide is indicated(underlined);

FIGS. 2A-2D illustrate the nucleotide sequence of the rat CHL gene (SEQID NO: 4) and the deduced amino acid sequence of rat CHL polypeptide(SEQ ID NO: 5). The predicted signal peptide is indicated (underlined);

FIGS. 3A-3C illustrate the nucleotide sequence of the human CHL gene(SEQ ID NO: 7) and the deduced amino acid sequence of human CHLpolypeptide (SEQ ID NO: 8). The predicted signal peptide is indicated(underlined and/or double-underlined);

FIGS. 4A-4C illustrate the nucleotide sequence of the human CHLd5 gene(SEQ ID NO: 11) and the deduced amino acid sequence of human CHLpolypeptide (SEQ ID NO: 12). The predicted signal peptide is indicated(underlined and/or double-underlined);

FIG. 5 illustrates the location of pro-collagen repeats (CR1-4) andpossible BMP1 cleavage sites (*) in murine CHL polypeptide and CHD/SOG;

FIGS. 6A-6E illustrate an amino acid sequence alignment of human CHLpolypeptide (Hchl; SEQ ID NO: 8), human CHLd5 polypeptide (Hchld5; SEQID NO: 12), murine CHL polypeptide (Mchl; SEQ ID NO: 2), rat CHLpolypeptide (Rchl; SEQ ID NO: 5), murine Chordin (Mchordin; SEQ ID NO:15), rat Chordin (Rchordin; SEQ ID NO: 16), and human Chordin (Hchordin;SEQ ID NO: 17);

FIGS. 7A-7B illustrate the expression of murine CHL mRNA and beta-actinas a control in (7A) adult tissues and (7B) embryos;

FIG. 8 illustrates the expression of human CHL mRNA and beta-actin as acontrol in adult and fetal human tissues;

FIGS. 9A-9B illustrate the differential expression of murine CHL mRNAand beta-actin as a control among the stroma cell lines OP9, D3, F4, andF10;

FIG. 10 illustrates the expression of murine CHL mRNA as detected by insitu hybridization in normal adult mouse lung, liver, stomach, andintestine (BV=blood vessel);

FIG. 11 illustrates the expression of murine CHL mRNA as detected by insitu hybridization in normal adult mouse kidney (boxed area is shown athigher magnification in the lower panel; C=cortex, M=medulla, P=papilla,and G=glomerulus);

FIG. 12 illustrates the expression of murine CHL mRNA as detected by insitu hybridization in normal embryonic and adult mouse brain;

FIG. 13 illustrates the expression of murine CHL mRNA as detected by insitu hybridization in normal embryonic and adult mouse bone;

FIGS. 14A-14C illustrate Western blot analysis of CHL-FLAG polypeptides;

FIGS. 15A-15B illustrate the secondary axis-forming activity of murineCHL polypeptide;

FIG. 16 illustrates the inhibition of Ter119+ erythroid cell generationfrom ES cells in vitro by the murine CHL-FLAG polypeptide.

FIG. 17 illustrates the results of a BMP-4-dependent cell proliferationand survival assay in which A5-F stromal cells were incubated withdifferent concentrations pf BMP-4 protein.

FIG. 18 illustrates the results of a BMP-4-dependent cell proliferationand survival assay in which A5-F stromal cells were incubated with aconstant concentration of BMP-4 protein and different cncentrations ofCHL polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited in this application are expressly incorporated byreference herein.

Definitions

The terms “CHL gene” or “CHL nucleic acid molecule” or “CHLpolynucleotide” refer to a nucleic acid molecule comprising orconsisting of a nucleotide sequence as set forth in any of SEQ ID NO: 1,SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 11, a nucleotide sequenceencoding the polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO:5, SEQ ID NO: 8, or SEQ ID NO: 12, a nucleotide sequence of the DNAinsert in any of ATCC Deposit Nos. PTA-961, PTA-962, PTA-963, orPTA-964, and nucleic acid molecules as defined herein.

The term “CHL polypeptide allelic variant” refers to one of severalpossible naturally occurring alternate forms of a gene occupying a givenlocus on a chromosome of an organism or a population of organisms.

The term “CHL polypeptide splice variant” refers to a nucleic acidmolecule, usually RNA, which is generated by alternative processing ofintron sequences in an RNA transcript of CHL polypeptide amino acidsequence as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:8, or SEQ ID NO: 12.

The term “isolated nucleic acid molecule” refers to a nucleic acidmolecule of the invention that (1) has been separated from at leastabout 50 percent of proteins, lipids, carbohydrates, or other materialswith which it is naturally found when total nucleic acid is isolatedfrom the source cells, (2) is not linked to all or a portion of apolynucleotide to which the “isolated nucleic acid molecule” is linkedin nature, (3) is operably linked to a polynucleotide which it is notlinked to in nature, or (4) does not occur in nature as part of a largerpolynucleotide sequence. Preferably, the isolated nucleic acid moleculeof the present invention is substantially free from any othercontaminating nucleic acid molecule(s) or other contaminants that arefound in its natural environment that would interfere with its use inpolypeptide production or its therapeutic, diagnostic, prophylactic orresearch use.

The term “nucleic acid sequence” or “nucleic acid molecule” refers to aDNA or RNA sequence. The term encompasses molecules formed from any ofthe known base analogs of DNA and RNA such as, but not limited to4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinyl-cytosine,pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “vector” is used to refer to any molecule (e.g., nucleic acid,plasmid, or virus) used to transfer coding information to a host cell.

The term “expression vector” refers to a vector that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control the expression of inserted heterologous nucleicacid sequences. Expression includes, but is not limited to, processessuch as transcription, translation, and RNA splicing, if introns arepresent.

The term “operably linked” is used herein to refer to an arrangement offlanking sequences wherein the flanking sequences so described areconfigured or assembled so as to perform their usual function. Thus, aflanking sequence operably linked to a coding sequence may be capable ofeffecting the replication, transcription and/or translation of thecoding sequence. For example, a coding sequence is operably linked to apromoter when the promoter is capable of directing transcription of thatcoding sequence. A flanking sequence need not be contiguous with thecoding sequence, so long as it functions correctly. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween a promoter sequence and the coding sequence and the promotersequence can still be considered “operably linked” to the codingsequence.

The term “host cell” is used to refer to a cell which has beentransformed, or is capable of being transformed with a nucleic acidsequence and then of expressing a selected gene of interest. The termincludes the progeny of the parent cell, whether or not the progeny isidentical in morphology or in genetic make-up to the original parent, solong as the selected gene is present.

The term “CHL polypeptide” refers to a polypeptide comprising the aminoacid sequence of any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQID NO: 12 and related polypeptides. Related polypeptides include CHLpolypeptide fragments, CHL polypeptide orthologs, CHL polypeptidevariants, and CHL polypeptide derivatives, which possess at least oneactivity of the polypeptide as set forth in any of SEQ ID NO: 2, SEQ IDNO: 5, SEQ ID NO: 8, or SEQ ID NO: 12. CHL polypeptides may be maturepolypeptides, as defined herein, and may or may not have anamino-terminal methionine residue, depending on the method by which theyare prepared.

The term “CHL polypeptide fragment” refers to a polypeptide thatcomprises a truncation at the amino-terminus (with or without a leadersequence) and/or a truncation at the carboxyl-terminus of thepolypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ IDNO: 8, or SEQ ID NO: 12. The term “CHL polypeptide fragment” also refersto amino-terminal and/or carboxyl-terminal truncations of CHLpolypeptide orthologs, CHL polypeptide derivatives, or CHL polypeptidevariants, or to amino-terminal and/or carboxyl-terminal truncations ofthe polypeptides encoded by CHL polypeptide allelic variants or CHLpolypeptide splice variants. CHL polypeptide fragments may result fromalternative RNA splicing or from in vivo protease activity.Membrane-bound forms of a CHL polypeptide are also contemplated by thepresent invention. In preferred embodiments, truncations and/ordeletions comprise about 10 amino acids, or about 20 amino acids, orabout 50 amino acids, or about 75 amino acids, or about 100 amino acids,or more than about 100 amino acids. The polypeptide fragments soproduced will comprise about 25 contiguous amino acids, or about 50amino acids, or about 75 amino acids, or about 100 amino acids, or about150 amino acids, or about 200 amino acids. Such CHL polypeptidefragments may optionally comprise an amino-terminal methionine residue.It will be appreciated that such fragments can be used, for example, togenerate antibodies to CHL polypeptides.

The term “CHL polypeptide ortholog” refers to a polypeptide from anotherspecies that corresponds to CHL polypeptide amino acid sequence as setforth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO:12. For example, mouse and human CHL polypeptides are consideredorthologs of each other.

The term “CHL polypeptide variants” refers to CHL polypeptidescomprising amino acid sequences having one or more amino acid sequencesubstitutions, deletions (such as internal deletions and/or CHLpolypeptide fragments), and/or additions (such as internal additionsand/or CHL fusion polypeptides) as compared to the CHL polypeptide aminoacid sequence set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:8, or SEQ ID NO: 12 (with or without a leader sequence). Variants may benaturally occurring (e.g., CHL polypeptide allelic variants, CHLpolypeptide orthologs, and CHL polypeptide splice variants) orartificially constructed. Such CHL polypeptide variants may be preparedfrom the corresponding nucleic acid molecules having a DNA sequence thatvaries accordingly from the DNA sequence as set forth in any of SEQ IDNO: 1, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 11. In preferredembodiments, the variants have from 1 to 3, or from 1 to 5, or from 1 to10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 50,or from 1 to 75, or from 1 to 100, or more than 100 amino acidsubstitutions, insertions, additions and/or deletions, wherein thesubstitutions may be conservative, or non-conservative, or anycombination thereof.

The term “CHL polypeptide derivatives” refers to the polypeptide as setforth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO:12, CHL polypeptide fragments, CHL polypeptide orthologs, or CHLpolypeptide variants, as defined herein, that have been chemicallymodified. The term “CHL polypeptide derivatives” also refers to thepolypeptides encoded by CHL polypeptide allelic variants or CHLpolypeptide splice variants, as defined herein, that have beenchemically modified.

The term “mature CHL polypeptide” refers to a CHL polypeptide lacking aleader sequence. A mature CHL polypeptide may also include othermodifications such as proteolytic processing of the amino-terminus (withor without a leader sequence) and/or the carboxyl-terminus, cleavage ofa smaller polypeptide from a larger precursor, N-linked and/or O-linkedglycosylation, and the like. Exemplary mature CHL polypeptides aredepicted by the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 6, SEQID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 14.

The term “CHL fusion polypeptide” refers to a fusion of one or moreamino acids (such as a heterologous protein or peptide) at the amino- orcarboxyl-terminus of the polypeptide as set forth in any of SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12, CHL polypeptidefragments, CHL polypeptide orthologs, CHL polypeptide variants, or CHLderivatives, as defined herein. The term “CHL fusion polypeptide” alsorefers to a fusion of one or more amino acids at the amino- orcarboxyl-terminus of the polypeptide encoded by CHL polypeptide allelicvariants or CHL polypeptide splice variants, as defined herein.

The term “biologically active CHL polypeptides” refers to CHLpolypeptides having at least one activity characteristic of thepolypeptide comprising the amino acid sequence of any of SEQ ID NO: 2,SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12. In addition, a CHLpolypeptide may be active as an immunogen; that is, the CHL polypeptidecontains at least one epitope to which antibodies may be raised.

The term “isolated polypeptide” refers to a polypeptide of the presentinvention that (1) has been separated from at least about 50 percent ofpolynucleotides, lipids, carbohydrates, or other materials with which itis naturally found when isolated from the source cell, (2) is not linked(by covalent or noncovalent interaction) to all or a portion of apolypeptide to which the “isolated polypeptide” is linked in nature, (3)is operably linked (by covalent or noncovalent interaction) to apolypeptide with which it is not linked in nature, or (4) does not occurin nature. Preferably, the isolated polypeptide is substantially freefrom any other contaminating polypeptides or other contaminants that arefound in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic or research use.

The term “identity,” as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequences.In the art, “identity” also means the degree of sequence relatednessbetween nucleic acid molecules or polypeptides, as the case may be, asdetermined by the match between strings of two or more nucleotide or twoor more amino acid sequences. “Identity” measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by a particular mathematical model orcomputer program (i.e., “algorithms”).

The term “similarity” is a related concept, but in contrast to“identity,” “similarity” refers to a measure of relatedness whichincludes both identical matches and conservative substitution matches.If two polypeptide sequences have, for example, 10/20 identical aminoacids, and the remainder are all non-conservative substitutions, thenthe percent identity and similarity would both be 50%. If in the sameexample, there are five more positions where there are conservativesubstitutions, then the percent identity remains 50%, but the percentsimilarity would be 75% (15/20). Therefore, in cases where there areconservative substitutions, the percent similarity between twopolypeptides will be higher than the percent identity between those twopolypeptides.

The term “naturally occurring” or “native” when used in connection withbiological materials such as nucleic acid molecules, polypeptides, hostcells, and the like, refers to materials which are found in nature andare not manipulated by man. Similarly, “non-naturally occurring” or“non-native” as used herein refers to a material that is not found innature or that has been structurally modified or synthesized by man.

The terms “effective amount” and “therapeutically effective amount” eachrefer to the amount of a CHL polypeptide or CHL nucleic acid moleculeused to support an observable level of one or more biological activitiesof the CHL polypeptides as set forth herein.

The term “pharmaceutically acceptable carrier” or “physiologicallyacceptable carrier” as used herein refers to one or more formulationmaterials suitable for accomplishing or enhancing the delivery of theCHL polypeptide, CHL nucleic acid molecule, or CHL selective bindingagent as a pharmaceutical composition.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

The term “selective binding agent” refers to a molecule or moleculeshaving specificity for a CHL polypeptide. As used herein, the terms,“specific” and “specificity” refer to the ability of the selectivebinding agents to bind to human CHL polypeptides and not to bind tohuman non-CHL polypeptides. It will be appreciated, however, that theselective binding agents may also bind orthologs of the polypeptide asset forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ IDNO: 12, that is, interspecies versions thereof, such as mouse and ratCHL polypeptides.

The term “transduction” is used to refer to the transfer of genes fromone bacterium to another, usually by a phage. “Transduction” also refersto the acquisition and transfer of eukaryotic cellular sequences byretroviruses.

The term “transfection” is used to refer to the uptake of foreign orexogenous DNA by a cell, and a cell has been “transfected” when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art and are disclosedherein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook etal., Molecular Cloning, A Laboratory Manual (Cold Spring HarborLaboratories, 1989); Davis et al., Basic Methods in Molecular Biology(Elsevier, 1986); and Chu et al., 1981, Gene 13:197. Such techniques canbe used to introduce one or more exogenous DNA moieties into suitablehost cells.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection or transduction, the transforming DNA may recombine withthat of the cell by physically integrating into a chromosome of thecell, may be maintained transiently as an episomal element without beingreplicated, or may replicate independently as a plasmid. A cell isconsidered to have been stably transformed when the DNA is replicatedwith the division of the cell.

Relatedness of Nucleic Acid Molecules and/or Polypeptides

It is understood that related nucleic acid molecules include allelic orsplice variants of the nucleic acid molecule of any of SEQ ID NO: 1, SEQID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 11, and include sequences whichare complementary to any of the above nucleotide sequences. Relatednucleic acid molecules also include a nucleotide sequence encoding apolypeptide comprising or consisting essentially of a substitution,modification, addition and/or deletion of one or more amino acidresidues compared to the polypeptide in any of SEQ ID NO: 2, SEQ ID NO:5, SEQ ID NO: 8, or SEQ ID NO: 12. Such related CHL polypeptides maycomprise, for example, an addition and/or a deletion of one or moreN-linked or O-linked glycosylation sites or an addition and/or adeletion of one or more cysteine residues.

Related nucleic acid molecules also include fragments of CHL nucleicacid molecules which encode a polypeptide of at least about 25contiguous amino acids, or about 50 amino acids, or about 75 aminoacids, or about 100 amino acids, or about 150 amino acids, or about 200amino acids, or more than 200 amino acid residues of the CHL polypeptideof any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12.

In addition, related CHL nucleic acid molecules also include thosemolecules which comprise nucleotide sequences which hybridize undermoderately or highly stringent conditions as defined herein with thefully complementary sequence of the CHL nucleic acid molecule of any ofSEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 11, or of amolecule encoding a polypeptide, which polypeptide comprises the aminoacid sequence as shown in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:8, or SEQ ID NO: 12, or of a nucleic acid fragment as defined herein, orof a nucleic acid fragment encoding a polypeptide as defined herein.Hybridization probes may be prepared using the CHL sequences providedherein to screen cDNA, genomic or synthetic DNA libraries for relatedsequences. Regions of the DNA and/or amino acid sequence of CHLpolypeptide that exhibit significant identity to known sequences arereadily determined using sequence alignment algorithms as describedherein and those regions may be used to design probes for screening.

The term “highly stringent conditions” refers to those conditions thatare designed to permit hybridization of DNA strands whose sequences arehighly complementary, and to exclude hybridization of significantlymismatched DNAs. Hybridization stringency is principally determined bytemperature, ionic strength, and the concentration of denaturing agentssuch as formamide. Examples of “highly stringent conditions” forhybridization and washing are 0.015 M sodium chloride, 0.0015 M sodiumcitrate at 65-68° C. or 0.015 M sodium chloride, 0.0015 M sodiumcitrate, and 50% formamide at 42° C. See Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring HarborLaboratory, 1989); Anderson et al., Nucleic Acid Hybridisation: APractical Approach Ch. 4 (IRL Press Limited).

More stringent conditions (such as higher temperature, lower ionicstrength, higher formamide, or other denaturing agent) may also beused—however, the rate of hybridization will be affected. Other agentsmay be included in the hybridization and washing buffers for the purposeof reducing non-specific and/or background hybridization. Examples are0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodiumpyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO₄, (SDS), ficoll,Denhardt's solution, sonicated salmon sperm DNA (or anothernon-complementary DNA), and dextran sulfate, although other suitableagents can also be used. The concentration and types of these additivescan be changed without substantially affecting the stringency of thehybridization conditions. Hybridization experiments are usually carriedout at pH 6.8-7.4; however, at typical ionic strength conditions, therate of hybridization is nearly independent of pH. See Anderson et al.,Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL PressLimited).

Factors affecting the stability of DNA duplex include base composition,length, and degree of base pair mismatch. Hybridization conditions canbe adjusted by one skilled in the art in order to accommodate thesevariables and allow DNAs of different sequence relatedness to formhybrids. The melting temperature of a perfectly matched DNA duplex canbe estimated by the following equation:T _(m)(°C.)=81.5+16.6(log [Na+])+0.41(% G+C)−600/N−0.72(% formamide)where N is the length of the duplex formed, [Na+] is the molarconcentration of the sodium ion in the hybridization or washingsolution, % G+C is the percentage of (guanine+cytosine) bases in thehybrid. For imperfectly matched hybrids, the melting temperature isreduced by approximately 1° C. for each 1% mismatch.

The term “moderately stringent conditions” refers to conditions underwhich a DNA duplex with a greater degree of base pair mismatching thancould occur under “highly stringent conditions” is able to form.Examples of typical “moderately stringent conditions” are 0.015 M sodiumchloride, 0.0015 M sodium citrate at 50-65° C. or 0.015 M sodiumchloride, 0.0015 M sodium citrate, and 20% formamide at 37-50° C. By wayof example, “moderately stringent conditions” of 50° C. in 0.015 Msodium ion will allow about a 21% mismatch.

It will be appreciated by those skilled in the art that there is noabsolute distinction between “highly stringent conditions” and“moderately stringent conditions.” For example, at 0.015 M sodium ion(no formamide), the melting temperature of perfectly matched long DNA isabout 71° C. With a wash at 65° C. (at the same ionic strength), thiswould allow for approximately a 6% mismatch. To capture more distantlyrelated sequences, one skilled in the art can simply lower thetemperature or raise the ionic strength.

A good estimate of the melting temperature in 1M NaCl* foroligonucleotide probes up to about 20nt is given by:T _(m)=2° C. per A-T base pair+4° C. per G-C base pair*The sodium ion concentration in 6× salt sodium citrate (SSC) is 1M. SeeSuggs et al., Developmental Biology Using Purified Genes 683 (Brown andFox, eds., 1981).

High stringency washing conditions for oligonucleotides are usually at atemperature of 0-5° C. below the Tm of the oligonucleotide in 6×SSC,0.1% SDS.

In another embodiment, related nucleic acid molecules comprise orconsist of a nucleotide sequence that is at least about 70 percentidentical to the nucleotide sequence as shown in any of SEQ ID NO: 1,SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 11, or comprise or consistessentially of a nucleotide sequence encoding a polypeptide that is atleast about 70 percent identical to the polypeptide as set forth in anyof SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12. Inpreferred embodiments, the nucleotide sequences are about 75 percent, orabout 80 percent, or about 85 percent, or about 90 percent, or about 95,96, 97, 98, or 99 percent identical to the nucleotide sequence as shownin any of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 11, orthe nucleotide sequences encode a polypeptide that is about 75 percent,or about 80 percent, or about 85 percent, or about 90 percent, or about95, 96, 97, 98, or 99 percent identical to the polypeptide sequence asset forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ IDNO: 12. Related nucleic acid molecules encode polypeptides possessing atleast one activity of the polypeptide set forth in any of SEQ ID NO: 2,SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12.

Differences in the nucleic acid sequence may result in conservativeand/or non-conservative modifications of the amino acid sequencerelative to the amino acid sequence of any of SEQ ID NO: 2, SEQ ID NO:5, SEQ ID NO: 8, or SEQ ID NO: 12.

Conservative modifications to the amino acid sequence of any of SEQ IDNO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 (and thecorresponding modifications to the encoding nucleotides) will produce apolypeptide having functional and chemical characteristics similar tothose of CHL polypeptides. In contrast, substantial modifications in thefunctional and/or chemical characteristics of CHL polypeptides may beaccomplished by selecting substitutions in the amino acid sequence ofany of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 thatdiffer significantly in their effect on maintaining (a) the structure ofthe molecular backbone in the area of the substitution, for example, asa sheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a nonnative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis.”

Conservative amino acid substitutions also encompass non-naturallyoccurring amino acid residues that are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics, and other reversed or invertedforms of amino acid moieties.

Naturally occurring residues may be divided into classes based on commonside chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr;

3) acidic: Asp, Glu;

4) basic: Asn, Gln, His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange ofa member of one of these classes for a member from another class. Suchsubstituted residues may be introduced into regions of the human CHLpolypeptide that are homologous with non-human CHL polypeptides, or intothe non-homologous regions of the molecule.

In making such changes, the hydropathic index of amino acids may beconsidered. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. The hydropathicindices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte et al., 1982, J. Mol. Biol. 157:105-31). It is known thatcertain amino acids may be substituted for other amino acids having asimilar hydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functionally equivalent protein orpeptide thereby created is intended for use in immunologicalembodiments, as in the present case. The greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine(−2.5); and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred. One may also identify epitopes from primaryamino acid sequences on the basis of hydrophilicity. These regions arealso referred to as “epitopic core regions.”

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the CHLpolypeptide, or to increase or decrease the affinity of the CHLpolypeptides described herein. Exemplary amino acid substitutions areset forth in Table I. TABLE I Amino Acid Substitutions OriginalExemplary Preferred Residues Substitutions Substitutions Ala Val, Leu,Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala SerGln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg IleLeu, Val, Met, Ala, Phe, Norleucine Leu Leu Norleucine, Ile, Val, Met,Ala, Phe Ile Lys Arg, 1,4 Diaminobutyric Acid, Gln, Asn Arg Met Leu,Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala,Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile,Met, Leu, Phe, Ala, Norleucine Leu

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ IDNO: 8, or SEQ ID NO: 12 using well-known techniques. For identifyingsuitable areas of the molecule that may be changed without destroyingbiological activity, one skilled in the art may target areas notbelieved to be important for activity. For example, when similarpolypeptides with similar activities from the same species or from otherspecies are known, one skilled in the art may compare the amino acidsequence of a CHL polypeptide to such similar polypeptides. With such acomparison, one can identify residues and portions of the molecules thatare conserved among similar polypeptides. It will be appreciated thatchanges in areas of the CHL molecule that are not conserved relative tosuch similar polypeptides would be less likely to adversely affect thebiological activity and/or structure of a CHL polypeptide. One skilledin the art would also know that, even in relatively conserved regions,one may substitute chemically similar amino acids for the naturallyoccurring residues while retaining activity (conservative amino acidresidue substitutions). Therefore, even areas that may be important forbiological activity or for structure may be subject to conservativeamino acid substitutions without destroying the biological activity orwithout adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a CHL polypeptide thatcorrespond to amino acid residues that are important for activity orstructure in similar polypeptides. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues of CHL polypeptides.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of CHL polypeptide withrespect to its three dimensional structure. One skilled in the art maychoose not to make radical changes to amino acid residues predicted tobe on the surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each amino acid residue. The variants could be screenedusing activity assays known to those with skill in the art. Suchvariants could be used to gather information about suitable variants.For example, if one discovered that a change to a particular amino acidresidue resulted in destroyed, undesirably reduced, or unsuitableactivity, variants with such a change would be avoided. In other words,based on information gathered from such routine experiments, one skilledin the art can readily determine the amino acids where furthersubstitutions should be avoided either alone or in combination withother mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult, 1996, Curr. Opin. Biotechnol.7:422-27; Chou et al., 1974, Biochemistry 13:22245; Chou et al., 1974,Biochemistry 113:211-22; Chou et al., 1978, Adv. Enzymol. Relat. AreasMol. Biol. 47:45-48; Chou et al., 1978, Ann. Rev. Biochem. 47:251-276;and Chou et al., 1979, Biophys. J. 26:367-84. Moreover, computerprograms are currently available to assist with predicting secondarystructure. One method of predicting secondary structure is based uponhomology modeling. For example, two polypeptides or proteins which havea sequence identity of greater than 30%, or similarity greater than 40%,often have similar structural topologies. The recent growth of theprotein structural database (PDB) has provided enhanced predictabilityof secondary structure, including the potential number of folds withinthe structure of a polypeptide or protein. See Holm et al., 1999,Nucleic Acids Res. 27:244-47. It has been suggested that there are alimited number of folds in a given polypeptide or protein and that oncea critical number of structures have been resolved, structuralprediction will become dramatically more accurate (Brenner et al., 1997,Curr. Opin. Struct. Biol. 7:369-76).

Additional methods of predicting secondary structure include “threading”(Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl et al., 1996,Structure 4:15-19), “profile analysis” (Bowie et al., 1991, Science,253:164-70; Gribskov et al., 1990, Methods Enzymol. 183:146-59; Gribskovet al, 1987, Proc. Nat. Acad. Sci. U.S.A. 84:4355-58), and “evolutionarylinkage” (See Holm et al., supra, and Brenner et al., supra).

Preferred CHL polypeptide variants include glycosylation variantswherein the number and/or type of glycosylation sites have been alteredcompared to the amino acid sequence set forth in any of SEQ ID NO: 2,SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12. In one embodiment, CHLpolypeptide variants comprise a greater or a lesser number of N-linkedglycosylation sites than the amino acid sequence set forth in any of SEQID NO: 2, SEQ ID NO: 5, SEQ ID NO:, 8, or SEQ ID NO: 12. An N-linkedglycosylation site is characterized by the sequence: Asn-X-Ser orAsn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionsthat eliminate this sequence will remove an existing N-linkedcarbohydrate chain. Also provided is a rearrangement of N-linkedcarbohydrate chains wherein one or more N-linked glycosylation sites(typically those that are naturally occurring) are eliminated and one ormore new N-linked sites are created. Additional preferred CHL variantsinclude cysteine variants, wherein one or more cysteine residues aredeleted or substituted with another amino acid (e.g., serine) ascompared to the amino acid sequence set forth in any of SEQ ID NO: 2,SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12. Cysteine variants areuseful when CHL polypeptides must be refolded into a biologically activeconformation such as after the isolation of insoluble inclusion bodies.Cysteine variants generally have fewer cysteine residues than the nativeprotein, and typically have an even number to minimize interactionsresulting from unpaired cysteines.

In other embodiments, related nucleic acid molecules comprise or consistof a nucleotide sequence encoding a polypeptide as set forth in any ofSEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12 with at leastone amino acid insertion and wherein the polypeptide has an activity ofthe polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ IDNO: 8, or SEQ ID NO: 12, or a nucleotide sequence encoding a polypeptideas set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQID NO: 12 with at least one amino acid deletion and wherein thepolypeptide has an activity of the polypeptide set forth in any of SEQID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12. Related nucleicacid molecules also comprise or consist of a nucleotide sequenceencoding a polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO:5, SEQ ID NO: 8, or SEQ ID NO: 12 wherein the polypeptide has acarboxyl- and/or amino-terminal truncation and further wherein thepolypeptide has an activity of the polypeptide set forth in any of SEQID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12. Related nucleicacid molecules also comprise or consist of a nucleotide sequenceencoding a polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO:5, SEQ ID NO: 8, or SEQ ID NO: 12 with at least one modificationselected from the group consisting of amino acid substitutions, aminoacid insertions, amino acid deletions, carboxyl-terminal truncations,and amino-terminal truncations and wherein the polypeptide has anactivity of the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO:5, SEQ ID NO: 8, or SEQ ID NO: 12.

In addition, the polypeptide comprising the amino acid sequence of anyof SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12, or otherCHL polypeptide, may be fused to a homologous polypeptide to form ahomodimer or to a heterologous polypeptide to form a heterodimer.Heterologous peptides and polypeptides include, but are not limited to:an epitope to allow for the detection and/or isolation of a CHL fusionpolypeptide; a transmembrane receptor protein or a portion thereof, suchas an extracellular domain or a transmembrane and intracellular domain;a ligand or a portion thereof which binds to a transmembrane receptorprotein; an enzyme or portion thereof which is catalytically active; apolypeptide or peptide which promotes oligomerization, such as a leucinezipper domain; a polypeptide or peptide which increases stability, suchas an immunoglobulin constant region; and a polypeptide which has atherapeutic activity different from the polypeptide comprising the aminoacid sequence as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ IDNO: 8, or SEQ ID NO: 12, or other CHL polypeptide.

Fusions can be made either at the amino-terminus or at thecarboxyl-terminus of the polypeptide comprising the amino acid sequenceset forth in any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ IDNO: 12, or other CHL polypeptide. Fusions may be direct with no linkeror adapter molecule or may be through a linker or adapter molecule. Alinker or adapter molecule may be one or more amino acid residues,typically from about 20 to about 50 amino acid residues. A linker oradapter molecule may also be designed with a cleavage site for a DNArestriction endonuclease or for a protease to allow for the separationof the fused moieties. It will be appreciated that once constructed, thefusion polypeptides can be derivatized according to the methodsdescribed herein.

In a further embodiment of the invention, the polypeptide comprising theamino acid sequence of any of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8,or SEQ ID NO: 12, or other CHL polypeptide, is fused to one or moredomains of an Fc region of human IgG. Antibodies comprise twofunctionally independent parts, a variable domain known as “Fab,” thatbinds an antigen, and a constant domain known as “Fc,” that is involvedin effector functions such as complement activation and attack byphagocytic cells. An Fc has a long serum half-life, whereas an Fab isshort-lived. Capon et al., 1989, Nature 337:525-31. When constructedtogether with a therapeutic protein, an Fc domain can provide longerhalf-life or incorporate such functions as Fc receptor binding, proteinA binding, complement fixation, and perhaps even placental transfer. Id.Table II summarizes the use of certain Fc fusions known in the art.TABLE II Fc Fusion with Therapeutic Proteins Form of Fc Fusion partnerTherapeutic implications Reference IgG1 N-terminus of Hodgkin's disease;anaplastic U.S. Pat. No. 5,480,981 CD30-L lymphoma; T-cell leukemiaMurine Fcγ2a IL-10 anti-inflammatory; transplant Zheng et al., 1995, J.rejection Immunol. 154: 5590-600 IgG1 TNF receptor septic shock Fisheret al., 1996, N. Engl. J. Med. 334: 1697-1702; Van Zee et al., 1996, J.Immunol. 156: 2221-30 IgG, IgA, IgM, or TNF receptor inflammation,autoimmune U.S. Pat. No. 5,808,029 IgE (excluding the disorders firstdomain) IgG1 CD4 receptor AIDS Capon et al., 1989, Nature 337: 525-31IgG1, N-terminus anti-cancer, antiviral Harvill et al., 1995, IgG3 ofIL-2 Immunotech. 1: 95-105 IgG1 C-terminus of OPG osteoarthritis; WO97/23614 bone density IgG1 N-terminus of leptin anti-obesity PCT/US97/23183, filed Dec. 11, 1997 Human Ig Cγ1 CTLA-4 autoimmune disordersLinsley, 1991, J. Exp. Med., 174: 561-69

In one example, a human IgG hinge, CH2, and CH3 region may be fused ateither the amino-terminus or carboxyl-terminus of the CHL polypeptidesusing methods known to the skilled artisan. In another example, a humanIgG hinge, CH2, and CH3 region may be fused at either the amino-terminusor carboxyl-terminus of a CHL polypeptide fragment (e.g., the predictedextracellular portion of CHL polypeptide).

The resulting CHL fusion polypeptide may be purified by use of a ProteinA affinity column. Peptides and proteins fused to an Fc region have beenfound to exhibit a substantially greater half-life in vivo than theunfused counterpart. Also, a fusion to an Fc region allows fordimerization/multimerization of the fusion polypeptide. The Fc regionmay be a naturally occurring Fc region, or may be altered to improvecertain qualities, such as therapeutic qualities, circulation time, orreduced aggregation.

Identity and similarity of related nucleic acid molecules andpolypeptides are readily calculated by known methods. Such methodsinclude, but are not limited to those described in ComputationalMolecular Biology (A. M. Lesk, ed., Oxford University Press 1988);Biocomputing: Informatics and Genome Projects (D. W. Smith, ed.,Academic Press 1993); Computer Analysis of Sequence Data (Part 1, A. M.Griffin and H. G. Griffin, eds., Humana Press 1994); G. von Heinle,Sequence Analysis in Molecular Biology (Academic Press 1987); SequenceAnalysis Primer (M. Gribskov and J. Devereux, eds., M. Stockton Press1991); and Carillo et al., 1988, SIAM J. Applied Math., 48:1073.

Preferred methods to determine identity and/or similarity are designedto give the largest match between the sequences tested. Methods todetermine identity and similarity are described in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux et al.,1984, Nucleic Acids Res. 12:387; Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,1990, J. Mol. Biol. 215:403-10). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (Altschul et al., BLAST Manual (NCB NLM NIH, Bethesda,Md.); Altschul et al., 1990, supra). The well-known Smith Watermanalgorithm may also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full-lengthsequences. Accordingly, in a preferred embodiment, the selectedalignment method (GAP program) will result in an alignment that spans atleast 50 contiguous amino acids of the claimed polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span,” asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3× the average diagonal; the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 0.1× the gap opening penalty), as well as a comparison matrixsuch as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.A standard comparison matrix is also used by the algorithm (see Dayhoffet al., 5 Atlas of Protein Sequence and Structure (Supp. 3 1978)(PAM250comparison matrix); Henikoff et al., 1992, Proc. Natl. Acad. Sci USA89:10915-19 (BLOSUM 62 comparison matrix)).

Preferred parameters for polypeptide sequence comparison include thefollowing:

Algorithm: Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-53;

Comparison matrix: BLOSUM 62 (Henikoffet al., supra);

Gap Penalty: 12

Gap Length Penalty: 4

Threshold of Similarity: 0

The GAP program is useful with the above parameters. The aforementionedparameters are the default parameters for polypeptide comparisons (alongwith no penalty for end gaps) using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparisoninclude the following:

Algorithm: Needleman and Wunsch, supra;

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

The GAP program is also useful with the above parameters. Theaforementioned parameters are the default parameters for nucleic acidmolecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, and thresholds of similarity may beused, including those set forth in the Program Manual, WisconsinPackage, Version 9, September, 1997. The particular choices to be madewill be apparent to those of skill in the art and will depend on thespecific comparison to be made, such as DNA-to-DNA, protein-to-protein,protein-to-DNA; and additionally, whether the comparison is betweengiven pairs of sequences (in which case GAP or BestFit are generallypreferred) or between one sequence and a large database of sequences (inwhich case FASTA or BLASTA are preferred).

Nucleic Acid Molecules

The nucleic acid molecules encoding a polypeptide comprising the aminoacid sequence of a CHL polypeptide can readily be obtained in a varietyof ways including, without limitation, chemical synthesis, cDNA orgenomic library screening, expression library screening, and/or PCRamplification of cDNA.

Recombinant DNA methods used herein are generally those set forth inSambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1989) and/or Current Protocols in MolecularBiology (Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons1994). The invention provides for nucleic acid molecules as describedherein and methods for obtaining such molecules.

Where a gene encoding the amino acid sequence of a CHL polypeptide hasbeen identified from one species, all or a portion of that gene may beused as a probe to identify orthologs or related genes from the samespecies. The probes or primers may be used to screen cDNA libraries fromvarious tissue sources believed to express the CHL polypeptide. Inaddition, part or all of a nucleic acid molecule having the sequence asset forth in any of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ IDNO: 11 may be used to screen a genomic library to identify and isolate agene encoding the amino acid sequence of a CHL polypeptide. Typically,conditions of moderate or high stringency will be employed for screeningto minimize the number of false positives obtained from the screening.

Nucleic acid molecules encoding the amino acid sequence of CHLpolypeptides may also be identified by expression cloning which employsthe detection of positive clones based upon a property of the expressedprotein. Typically, nucleic acid libraries are screened by the bindingan antibody or other binding partner (e.g., receptor or ligand) tocloned proteins that are expressed and displayed on a host cell surface.The antibody or binding partner is modified with a detectable label toidentify those cells expressing the desired clone.

Recombinant expression techniques conducted in accordance with thedescriptions set forth below may be followed to produce thesepolynucleotides and to express the encoded polypeptides. For example, byinserting a nucleic acid sequence that encodes the amino acid sequenceof a CHL polypeptide into an appropriate vector, one skilled in the artcan readily produce large quantities of the desired nucleotide sequence.The sequences can then be used to generate detection probes oramplification primers. Alternatively, a polynucleotide encoding theamino acid sequence of a CHL polypeptide can be inserted into anexpression vector. By introducing the expression vector into anappropriate host, the encoded CHL polypeptide may be produced in largeamounts.

Another method for obtaining a suitable nucleic acid sequence is thepolymerase chain reaction (PCR). In this method, cDNA is prepared frompoly(A)+RNA or total RNA using the enzyme reverse transcriptase. Twoprimers, typically complementary to two separate regions of cDNAencoding the amino acid sequence of a CHL polypeptide, are then added tothe cDNA along with a polymerase such as Taq polymerase, and thepolymerase amplifies the cDNA region between the two primers.

Another means of preparing a nucleic acid molecule encoding the aminoacid sequence of a CHL polypeptide is chemical synthesis using methodswell known to the skilled artisan such as those described by Engels etal., 1989, Angew. Chem. Intl. Ed. 28:716-34. These methods include,inter alia, the phosphotriester, phosphoramidite, and H-phosphonatemethods for nucleic acid synthesis. A preferred method for such chemicalsynthesis is polymer-supported synthesis using standard phosphoramiditechemistry. Typically, the DNA encoding the amino acid sequence of a CHLpolypeptide will be several hundred nucleotides in length. Nucleic acidslarger than about 100 nucleotides can be synthesized as severalfragments using these methods. The fragments can then be ligatedtogether to form the full-length nucleotide sequence of a CHL gene.Usually, the DNA fragment encoding the amino-terminus of the polypeptidewill have an ATG, which encodes a methionine residue. This methioninemay or may not be present on the mature form of the CHL polypeptide,depending on whether the polypeptide produced in the host cell isdesigned to be secreted from that cell. Other methods known to theskilled artisan may be used as well.

In certain embodiments, nucleic acid variants contain codons which havebeen altered for optimal expression of a CHL polypeptide in a given hostcell. Particular codon alterations will depend upon the CHL polypeptideand host cell selected for expression. Such “codon optimization” can becarried out by a variety of methods, for example, by selecting codonswhich are preferred for use in highly expressed genes in a given hostcell. Computer algorithms which incorporate codon frequency tables suchas “Eco_high.Cod” for codon preference of highly expressed bacterialgenes may be used and are provided by the University of WisconsinPackage Version 9.0 (Genetics Computer Group, Madison, Wis.). Otheruseful codon frequency tables include “Celegans_high.cod,”“Celegans_low.cod,” “Drosophila_high.cod,” “Human_high.cod,”“Maize_high.cod,” and “Yeast_high.cod.”

In some cases, it may be desirable to prepare nucleic acid moleculesencoding CHL polypeptide variants. Nucleic acid molecules encodingvariants may be produced using site directed mutagenesis, PCRamplification, or other appropriate methods, where the primer(s) havethe desired point mutations (see Sambrook et al., supra, and Ausubel etal., supra, for descriptions of mutagenesis techniques). Chemicalsynthesis using methods described by Engels et al., supra, may also beused to prepare such variants. Other methods known to the skilledartisan may be used as well.

Vectors and Host Cells

A nucleic acid molecule encoding the amino acid sequence of a CHLpolypeptide is inserted into an appropriate expression vector usingstandard ligation techniques. The vector is typically selected to befunctional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene can occur). A nucleic acid moleculeencoding the amino acid sequence of a CHL polypeptide may beamplified/expressed in prokaryotic, yeast, insect (baculovirus systems)and/or eukaryotic host cells. Selection of the host cell will depend inpart on whether a CHL polypeptide is to be post-translationally modified(e.g., glycosylated and/or phosphorylated). If so, yeast, insect, ormammalian host cells are preferable. For a review of expression vectors,see Meth. Enz., vol. 185 (D. V. Goeddel, ed., Academic Press 1990).

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the CHLpolypeptide coding sequence; the oligonucleotide sequence encodespolyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus), or myc for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification of the CHL polypeptide from the host cell. Affinitypurification can be accomplished, for example, by column chromatographyusing antibodies against the tag as an affinity matrix. Optionally, thetag can subsequently be removed from the purified CHL polypeptide byvarious means such as using certain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), or synthetic, or theflanking sequences may be native sequences which normally function toregulate CHL polypeptide expression. As such, the source of a flankingsequence may be any prokaryotic or eukaryotic organism, any vertebrateor invertebrate organism, or any plant, provided that the flankingsequence is functional in, and can be activated by, the host cellmachinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein—other than the CHL gene flankingsequences—will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of a flanking sequence may beknown. Here, the flanking sequence may be synthesized using the methodsdescribed herein for nucleic acid synthesis or cloning.

Where all or only a portion of the flanking sequence is known, it may beobtained using PCR and/or by screening a genomic library with a suitableoligonucleofide and/or flanking sequence fragment from the same oranother species. Where the flanking sequence is not known, a fragment ofDNA containing a flanking sequence may be isolated from a larger pieceof DNA that may contain, for example, a coding sequence or even anothergene or genes. Isolation may be accomplished by restriction endonucleasedigestion to produce the proper DNA fragment followed by isolation usingagarose gel purification, Qiagen® column chromatography (Chatsworth,Calif.), or other methods known to the skilled artisan. The selection ofsuitable enzymes to accomplish this purpose will be readily apparent toone of ordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. Amplification of the vectorto a certain copy number can, in some cases, be important for theoptimal expression of a CHL polypeptide. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria andvarious origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitusvirus (VSV), or papillomaviruses such as HPV or BPV) are useful forcloning vectors in mammalian cells. Generally, the origin of replicationcomponent is not needed for mammalian expression vectors (for example,the SV40 origin is often used only because it contains the earlypromoter).

A transcription termination sequence is typically located 3′ of the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells; (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A neomycin resistance gene may also beused for selection in prokaryotic and eukaryotic host cells.

Other selection genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and thymidine kinase. Themammalian cell transformants are placed under selection pressure whereinonly the transformants are uniquely adapted to survive by virtue of theselection gene present in the vector. Selection pressure is imposed byculturing the transformed cells under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to the amplification of both the selection gene and theDNA that encodes a CHL polypeptide. As a result, increased quantities ofCHL polypeptide are synthesized from the amplified DNA.

A ribosome binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of a CHL polypeptide to beexpressed. The Shine-Dalgarno sequence is varied but is typically apolypurine (i.e., having a high A-G content). Many Shine-Dalgarnosequences have been identified, each of which can be readily synthesizedusing methods set forth herein and used in a prokaryotic vector.

A leader, or signal, sequence may be used to direct a CHL polypeptideout of the host cell. Typically, a nucleotide sequence encoding thesignal sequence is positioned in the coding region of a CHL nucleic acidmolecule, or directly at the 5′ end of a CHL polypeptide coding region.Many signal sequences have been identified, and any of those that arefunctional in the selected host cell may be used in conjunction with aCHL nucleic acid molecule. Therefore, a signal sequence may behomologous (naturally occurring) or heterologous to the CHL nucleic acidmolecule. Additionally, a signal sequence may be chemically synthesizedusing methods described herein. In most cases, the secretion of a CHLpolypeptide from the host cell via the presence of a signal peptide willresult in the removal of the signal peptide from the secreted CHLpolypeptide. The signal sequence may be a component of the vector, or itmay be a part of a CHL nucleic acid molecule that is inserted into thevector.

Included within the scope of this invention is the use of either anucleotide sequence encoding a native CHL polypeptide signal sequencejoined to a CHL polypeptide coding region or a nucleotide sequenceencoding a heterologous signal sequence joined to a CHL polypeptidecoding region. The heterologous signal sequence selected should be onethat is recognized and processed, i.e., cleaved by a signal peptidase,by the host cell. For prokaryotic host cells that do not recognize andprocess the native CHL polypeptide signal sequence, the signal sequenceis substituted by a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, orheat-stable enterotoxin II leaders. For yeast secretion, the native CHLpolypeptide signal sequence may be substituted by the yeast invertase,alpha factor, or acid phosphatase leaders. In mammalian cell expressionthe native signal sequence is satisfactory, although other mammaliansignal sequences may be suitable.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various presequencesto improve glycosylation or yield. For example, one may alter thepeptidase cleavage site of a particular signal peptide, or addpro-sequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired CHL polypeptide, if the enzymecuts at such area within the mature polypeptide.

In many cases, transcription of a nucleic acid molecule is increased bythe presence of one or more introns in the vector; this is particularlytrue where a polypeptide is produced in eukaryotic host cells,especially mammalian host cells. The introns used may be naturallyoccurring within the CHL gene especially where the gene used is afull-length genomic sequence or a fragment thereof. Where the intron isnot naturally occurring within the gene (as for most cDNAs), the intronmay be obtained from another source. The position of the intron withrespect to flanking sequences and the CHL gene is generally important,as the intron must be transcribed to be effective. Thus, when a CHL cDNAmolecule is being transcribed, the preferred position for the intron is3′ to the transcription start site and 5′ to the poly-A transcriptiontermination sequence. Preferably, the intron or introns will be locatedon one side or the other (i.e., 5′ or 3′) of the cDNA such that it doesnot interrupt the coding sequence. Any intron from any source, includingviral, prokaryotic and eukaryotic (plant or animal) organisms, may beused to practice this invention, provided that it is compatible with thehost cell into which it is inserted. Also included herein are syntheticintrons. Optionally, more than one intron may be used in the vector.

The expression and cloning vectors of the present invention willtypically contain a promoter that is recognized by the host organism andoperably linked to the molecule encoding the CHL polypeptide. Promotersare untranscribed sequences located upstream (i.e., 5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,initiate continual gene product production; that is, there is little orno control over gene expression. A large number of promoters, recognizedby a variety of potential host cells, are well known. A suitablepromoter is operably linked to the DNA encoding CHL polypeptide byremoving the promoter from the source DNA by restriction enzymedigestion and inserting the desired promoter sequence into the vector.The native CHL promoter sequence may be used to direct amplificationand/or expression of a CHL nucleic acid molecule. A heterologouspromoter is preferred, however, if it permits greater transcription andhigher yields of the expressed protein as compared to the nativepromoter, and if it is compatible with the host cell system that hasbeen selected for use.

Promoters suitable for use with prokaryotic hosts include thebeta-lactamase and lactose promoter systems; alkaline phosphatase; atryptophan (trp) promoter system; and hybrid promoters such as the tacpromoter. Other known bacterial promoters are also suitable. Theirsequences have been published, thereby enabling one skilled in the artto ligate them to the desired DNA sequence, using linkers or adapters asneeded to supply any useful restriction sites.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest in controlling CHL geneexpression include, but are not limited to: the SV40 early promoterregion (Bernoist and Chambon, 1981, Nature 290:304-10); the CMVpromoter; the promoter contained in the 3′ long terminal repeat of Roussarcoma virus (Yamamoto, et al., 1980, Cell 22:787-97); the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1444-45); the regulatory sequences of the metallothionine gene(Brinster et al., 1982, Nature 296:39-42); prokaryotic expressionvectors such as the beta-lactamase promoter (Villa-Kamaroff et al.,1978, Proc. Nati. Acad. Sci. U.S.A., 75:3727-31); or the tac promoter(DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80:21-25). Also ofinterest are the following animal transcriptional control regions, whichexhibit tissue specificity and have been utilized in transgenic animals:the elastase I gene control region which is active in pancreatic acinarcells (Swift et al., 1984, Cell 38:639-46; Omitz et al., 1986, ColdSpring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, 1987,Hepatology 7:425-515); the insulin gene control region which is activein pancreatic beta cells (Hanahan, 1985, Nature 315:115-22); theimmunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-58; Adames et al., 1985, Nature318:533-38; Alexander et al., 1987, Mol. Cell. Biol., 7:1436-44); themouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-95);the albumin gene control region which is active in liver (Pinkert etal., 1987, Genes and Devel. 1:268-76); the alpha-feto-protein genecontrol region which is active in liver (Krumlauf et al., 1985, Mol.Cell. Biol., 5:1639-48; Hammer et al., 1987, Science 235:53-58); thealpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., 1987, Genes and Devel. 1:161-71); the beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-40; Kollias et al., 1986, Cell 46:89-94); the myelinbasic protein gene control region which is active in oligodendrocytecells in the brain (Readhead et al., 1987, Cell 48:703-12); the myosinlight chain-2 gene control region which is active in skeletal muscle(Sani, 1985, Nature 314:283-86); and the gonadotropic releasing hormonegene control region which is active in the hypothalamus (Mason et al.,1986, Science 234:1372-78).

An enhancer sequence may be inserted into the vector to increase thetranscription of a DNA encoding a CHL polypeptide of the presentinvention by higher eukaryotes. Enhancers are cis-acting elements ofDNA, usually about 10-300 bp in length, that act on the promoter toincrease transcription. Enhancers are relatively orientation andposition independent. They have been found 5′ and 3′ to thetranscription unit. Several enhancer sequences available from mammaliangenes are known (e.g., globin, elastase, albumin, alpha-feto-protein andinsulin). Typically, however, an enhancer from a virus will be used. TheSV40 enhancer, the cytomegalovirus early promoter enhancer, the polyomaenhancer, and adenovirus enhancers are exemplary enhancing elements forthe activation of eukaryotic promoters. While an enhancer may be splicedinto the vector at a position 5′ or 3′ to a CHL nucleic acid molecule,it is typically located at a site 5′ from the promoter.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences described herein are not already present in thevector, they may be individually obtained and ligated into the vector.Methods used for obtaining each of the flanking sequences are well knownto one skilled in the art.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen, SanDiego, Calif.), pBSII (Stratagene, La Jolla, Calif.), pET15 (Novagen,Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2(Clontech, Palo Alto, Calif.), pETL (BlueBacII, Invitrogen), pDSR-alpha(PCT Pub. No. WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island,N.Y.).

Additional suitable vectors include, but are not limited to, cosmids,plasmids, or modified viruses, but it will be appreciated that thevector system must be compatible with the selected host cell. Suchvectors include, but are not limited to plasmids such as Bluescript®plasmid derivatives (a high copy number ColE1-based phagemid, StratageneCloning Systems, La Jolla Calif.), PCR cloning plasmids designed forcloning Taq-amplified PCR products (e.g., TOPO™ TA Cloning® Kit, PCR2.1®plasmid derivatives, Invitrogen, Carlsbad, Calif.), and mammalian, yeastor virus vectors such as a baculovirus expression system (pBacPAKplasmid derivatives, Clontech, Palo Alto, Calif.).

After the vector has been constructed and a nucleic acid moleculeencoding a CHL polypeptide has been inserted into the proper site of thevector, the completed vector may be inserted into a suitable host cellfor amplification and/or polypeptide expression. The transformation ofan expression vector for a CHL polypeptide into a selected host cell maybe accomplished by well known methods including methods such astransfection, infection, calcium chloride, electroporation,microinjection, lipofection, DEAE-dextran method, or other knowntechniques. The method selected will in part be a function of the typeof host cell to be used. These methods and other suitable methods arewell known to the skilled artisan, and are set forth, for example, inSambrook et al., supra.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (such as a yeast, insect, or vertebrate cell). The host cell,when cultured under appropriate conditions, synthesizes a CHLpolypeptide which can subsequently be collected from the culture medium(if the host cell secretes it into the medium) or directly from the hostcell producing it (if it is not secreted). The selection of anappropriate host cell will depend upon various factors, such as desiredexpression levels, polypeptide modifications that are desirable ornecessary for activity (such as glycosylation or phosphorylation) andease of folding into a biologically active molecule.

A number of suitable host cells are known in the art and many areavailable from the American Type Culture Collection (ATCC), Manassas,Va. Examples include, but are not limited to, mammalian cells, such asChinese hamster ovary cells (CHO), CHO DHFR(−) cells (Urlaub et al.,1980, Proc. Natl. Acad. Sci. U.S.A. 97:4216-20), human embryonic kidney(HEK) 293 or 293T cells, or 3T3 cells. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening, product production, and purification are knownin the art. Other suitable mammalian cell lines, are the monkey COS-1and COS-7 cell lines, and the CV-1 cell line. Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells may be genotypicallydeficient in the selection gene, or may contain a dominantly actingselection gene. Other suitable mammalian cell lines include but are notlimited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster celllines. Each of these cell lines is known by and available to thoseskilled in the art of protein expression.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5□, DH10, and MC1061) are well-known as host cells in the fieldof biotechnology. Various strains of B. subtilis, Pseudomonas spp.,other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for the expression of the polypeptides of thepresent invention. Preferred yeast cells include, for example,Saccharomyces cerivisae and Pichia pastoris.

Additionally, where desired, insect cell systems may be utilized in themethods of the present invention. Such systems are described, forexample, in Kitts et al., 1993, Biotechniques, 14:810-17; Lucklow, 1993,Curr. Opin. Biotechnol. 4:564-72; and Lucklow et al., 1993, J. Virol.,67:4566-79. Preferred insect cells are Sf-9 and Hi5 (Invitrogen).

One may also use transgenic animals to express glycosylated CHLpolypeptides. For example, one may use a transgenic milk-producinganimal (a cow or goat, for example) and obtain the present glycosylatedpolypeptide in the animal milk. One may also use plants to produce CHLpolypeptides, however, in general, the glycosylation occurring in plantsis different from that produced in mammalian cells, and may result in aglycosylated product which is not suitable for human therapeutic use.

Polypeptide Production

Host cells comprising a CHL polypeptide expression vector may becultured using standard media well known to the skilled artisan. Themedia will usually contain all nutrients necessary for the growth andsurvival of the cells. Suitable media for culturing E. coli cellsinclude, for example, Luria Broth (LB) and/or Terrific Broth (TB).Suitable media for culturing eukaryotic cells include Roswell ParkMemorial Institute medium 1640 (RPMI 1640), Minimal Essential Medium(MEM) and/or Dulbecco's Modified Eagle Medium (DMEM), all of which maybe supplemented with serum and/or growth factors as necessary for theparticular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary.

Typically, an antibiotic or other compound useful for selective growthof transfected or transformed cells is added as a supplement to themedia. The compound to be used will be dictated by the selectable markerelement present on the plasmid with which the host cell was transformed.For example, where the selectable marker element is kanamycinresistance, the compound added to the culture medium will be kanamycin.Other compounds for selective growth include ampicillin, tetracycline,and neomycin.

The amount of a CHL polypeptide produced by a host cell can be evaluatedusing standard methods known in the art. Such methods include, withoutlimitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, High PerformanceLiquid Chromatography (HPLC) separation, immunoprecipitation, and/oractivity assays such as DNA binding gel shift assays.

If a CHL polypeptide has been designed to be secreted from the hostcells, the majority of polypeptide may be found in the cell culturemedium. If however, the CHL polypeptide is not secreted from the hostcells, it will be present in the cytoplasm and/or the nucleus (foreukaryotic host cells) or in the cytosol (for gram-negative bacteriahost cells).

For a CHL polypeptide situated in the host cell cytoplasm and/or nucleus(for eukaryotic host cells) or in the cytosol (for bacterial hostcells), the intracellular material (including inclusion bodies forgram-negative bacteria) can be extracted from the host cell using anystandard technique known to the skilled artisan. For example, the hostcells can be lysed to release the contents of the periplasm/cytoplasm byFrench press, homogenization, and/or sonication followed bycentrifugation.

If a CHL polypeptide has formed inclusion bodies in the cytosol, theinclusion bodies can often bind to the inner and/or outer cellularmembranes and thus will be found primarily in the pellet material aftercentrifugation. The pellet material can then be treated at pH extremesor with a chaotropic agent such as a detergent, guanidine, guanidinederivatives, urea, or urea derivatives in the presence of a reducingagent such as dithiothreitol at alkaline pH or tris carboxyethylphosphine at acid pH to release, break apart, and solubilize theinclusion bodies. The solubilized CHL polypeptide can then be analyzedusing gel electrophoresis, immunoprecipitation, or the like. If it isdesired to isolate the CHL polypeptide, isolation may be accomplishedusing standard methods such as those described herein and in Marston etal., 1990, Meth. Enz., 182:264-75.

In some cases, a CHL polypeptide may not be biologically active uponisolation. Various methods for “refolding” or converting the polypeptideto its tertiary structure and generating disulfide linkages can be usedto restore biological activity. Such methods include exposing thesolubilized polypeptide to a pH usually above 7 and in the presence of aparticular concentration of a chaotrope. The selection of chaotrope isvery similar to the choices used for inclusion body solubilization, butusually the chaotrope is used at a lower concentration and is notnecessarily the same as chaotropes used for the solubilization. In mostcases the refolding/oxidation solution will also contain a reducingagent or the reducing agent plus its oxidized form in a specific ratioto generate a particular redox potential allowing for disulfideshuffling to occur in the formation of the protein's cysteine bridges.Some of the commonly used redox couples include cysteine/cystamine,glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol(DTT)/dithiane DTT, and2-2-mercaptoethanol(bME)/dithio-b(ME). In many instances, a cosolventmay be used or may be needed to increase the efficiency of therefolding, and the more common reagents used for this purpose includeglycerol, polyethylene glycol of various molecular weights, arginine andthe like.

If inclusion bodies are not formed to a significant degree uponexpression of a CHL polypeptide, then the polypeptide will be foundprimarily in the supematant after centrifugation of the cell homogenate.The polypeptide may be further isolated from the supernatant usingmethods such as those described herein.

The purification of a CHL polypeptide from solution can be accomplishedusing a variety of techniques. If the polypeptide has been synthesizedsuch that it contains a tag such as Hexahistidine (CHLpolypeptide/hexaHis) or other small peptide such as FLAG (Eastman KodakCo., New Haven, Conn.) or myc (Invitrogen, Carlsbad, Calif.) at eitherits carboxyl- or amino-terminus, it may be purified in a one-stepprocess by passing the solution through an affinity column where thecolumn matrix has a high affinity for the tag.

For example, polyhistidine binds with great affinity and specificity tonickel. Thus, an affinity column of nickel (such as the Qiagen® nickelcolumns) can be used for purification of CHL polypeptide/polyHis. See,e.g., Current Protocols in Molecular Biology § 10.11.8 (Ausubel et al.,eds., Green Publishers Inc. and Wiley and Sons 1993).

Additionally, CHL polypeptides may be purified through the use of amonoclonal antibody that is capable of specifically recognizing andbinding to a CHL polypeptide.

Other suitable procedures for purification include, without limitation,affinity chromatography, immunoaffinity chromatography, ion exchangechromatography, molecular sieve chromatography, HPLC, electrophoresis(including native gel electrophoresis) followed by gel elution, andpreparative isoelectric focusing (“Isoprime” machine/technique, HoeferScientific, San Francisco, Calif.). In some cases, two or morepurification techniques may be combined to achieve increased purity.

CHL polypeptides may also be prepared by chemical synthesis methods(such as solid phase peptide synthesis) using techniques known in theart such as those set forth by Merrifield et al., 1963, J. Am. Chem.Soc. 85:2149; Houghten et al., 1985, Proc Natl Acad. Sci. USA 82:5132;and Stewart and Young, Solid Phase Peptide Synthesis (Pierce ChemicalCo. 1984). Such polypeptides may be synthesized with or without amethionine on the amino-terminus. Chemically synthesized CHLpolypeptides may be oxidized using methods set forth in these referencesto form disulfide bridges. Chemically synthesized CHL polypeptides areexpected to have comparable biological activity to the corresponding CHLpolypeptides produced recombinantly or purified from natural sources,and thus may be used interchangeably with a recombinant or natural CHLpolypeptide.

Another means of obtaining CHL polypeptide is via purification frombiological samples such as source tissues and/or fluids in which the CHLpolypeptide is naturally found. Such purification can be conducted usingmethods for protein purification as described herein. The presence ofthe CHL polypeptide during purification may be monitored, for example,using an antibody prepared against recombinantly produced CHLpolypeptide or peptide fragments thereof.

A number of additional methods for producing nucleic acids andpolypeptides are known in the art, and the methods can be used toproduce polypeptides having specificity for CHL polypeptide. See, e.g.,Roberts et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:12297-303, whichdescribes the production of fusion proteins between an mRNA and itsencoded peptide. See also, Roberts, 1999, Curr. Opin. Chem. Biol.3:268-73. Additionally, U.S. Pat. No. 5,824,469 describes methods forobtaining oligonucleotides capable of carrying out a specific biologicalfunction. The procedure involves generating a heterogeneous pool ofoligonucleotides, each having a 5′ randomized sequence, a centralpreselected sequence, and a 3′ randomized sequence. The resultingheterogeneous pool is introduced into a population of cells that do notexhibit the desired biological function. Subpopulations of the cells arethen screened for those that exhibit a predetermined biologicalfunction. From that subpopulation, oligonucleotides capable of carryingout the desired biological function are isolated.

U.S. Pat. Nos. 5,763,192; 5,814,476; 5,723,323; and 5,817,483 describeprocesses for producing peptides or polypeptides. This is done byproducing stochastic genes or fragments thereof, and then introducingthese genes into host cells which produce one or more proteins encodedby the stochastic genes. The host cells are then screened to identifythose clones producing peptides or polypeptides having the desiredactivity.

Another method for producing peptides or polypeptides is described inPCT/US98/20094 (WO99/15650) filed by Athersys, Inc. Known as “RandomActivation of Gene Expression for Gene Discovery” (RAGE-GD), the processinvolves the activation of endogenous gene expression or over-expressionof a gene by in situ recombination methods. For example, expression ofan endogenous gene is activated or increased by integrating a regulatorysequence into the target cell which is capable of activating expressionof the gene by non-homologous or illegitimate recombination. The targetDNA is first subjected to radiation, and a genetic promoter inserted.The promoter eventually locates a break at the front of a gene,initiating transcription of the gene. This results in expression of thedesired peptide or polypeptide.

It will be appreciated that these methods can also be used to createcomprehensive CHL polypeptide expression libraries, which cansubsequently be used for high throughput phenotypic screening in avariety of assays, such as biochemical assays, cellular assays, andwhole organism assays (e.g., plant, mouse, etc.).

Synthesis

It will be appreciated by those skilled in the art that the nucleic acidand polypeptide molecules described herein may be produced byrecombinant and other means.

Selective Binding Agents

The term “selective binding agent” refers to a molecule that hasspecificity for one or more CHL polypeptides. Suitable selective bindingagents include, but are not limited to, antibodies and derivativesthereof, polypeptides, and small molecules. Suitable selective bindingagents may be prepared using methods known in the art. An exemplary CHLpolypeptide selective binding agent of the present invention is capableof binding a certain portion of the CHL polypeptide thereby inhibitingthe binding of the polypeptide to a CHL polypeptide receptor.

Selective binding agents such as antibodies and antibody fragments thatbind CHL polypeptides are within the scope of the present invention. Theantibodies may be polyclonal including monospecific polyclonal;monoclonal (MAbs); recombinant; chimeric; humanized, such asCDR-grafted; human; single chain; and/or bispecific; as well asfragments; variants; or derivatives thereof. Antibody fragments includethose portions of the antibody that bind to an epitope on the CHLpolypeptide. Examples of such fragments include Fab and F(ab′) fragmentsgenerated by enzymatic cleavage of full-length antibodies. Other bindingfragments include those generated by recombinant DNA techniques, such asthe expression of recombinant plasmids containing nucleic acid sequencesencoding antibody variable regions.

Polyclonal antibodies directed toward a CHL polypeptide generally areproduced in animals (e.g., rabbits or mice) by means of multiplesubcutaneous or intraperitoneal injections of CHL polypeptide and anadjuvant. It may be useful to conjugate a CHL polypeptide to a carrierprotein that is immunogenic in the species to be immunized, such askeyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, orsoybean trypsin inhibitor. Also, aggregating agents such as alum areused to enhance the immune response. After immunization, the animals arebled and the serum is assayed for anti-CHL antibody titer.

Monoclonal antibodies directed toward CHL polypeptides are producedusing any method that provides for the production of antibody moleculesby continuous cell lines in culture. Examples of suitable methods forpreparing monoclonal antibodies include the hybridoma methods of Kohleret al., 1975, Nature 256:495-97 and the human B-cell hybridoma method(Kozbor, 1984, J. Immunol. 133:3001; Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications 51-63 (Marcel Dekker, Inc.,1987). Also provided by the invention are hybridoma cell lines thatproduce monoclonal antibodies reactive with CHL polypeptides.

Monoclonal antibodies of the invention may be modified for use astherapeutics. One embodiment is a “chimeric” antibody in which a portionof the heavy (H) and/or light (L) chain is identical with or homologousto a corresponding sequence in antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remainder of the chain(s) is/are identical with or homologous to acorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass. Also included arefragments of such antibodies, so long as they exhibit the desiredbiological activity. See U.S. Pat. No. 4,816,567; Morrison et al., 1985,Proc. Natl. Acad. Sci. 81:6851-55.

In another embodiment, a monoclonal antibody of the invention is a“humanized” antibody. Methods for humanizing non-human antibodies arewell known in the art. See U.S. Pat. Nos. 5,585,089 and 5,693,762.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. Humanization can beperformed, for example, using methods described in the art (Jones etal., 1986, Nature 321:522-25; Riechmann et al., 1998, Nature 332:323-27;Verhoeyen et al., 1988, Science 239:1534-36), by substituting at least aportion of a rodent complementarity-determining region (CDR) for thecorresponding regions of a human antibody.

Also encompassed by the invention are human antibodies that bind CHLpolypeptides. Using transgenic animals (e.g., mice) that are capable ofproducing a repertoire of human antibodies in the absence of endogenousimmunoglobulin production such antibodies are produced by immunizationwith a CHL polypeptide antigen (i.e., having at least 6 contiguous aminoacids), optionally conjugated to a carrier. See, e.g., Jakobovits etal., 1993, Proc. Natl. Acad. Sci. 90:2551-55; Jakobovits et al., 1993,Nature 362:255-58; Bruggermann et al., 1993, Year in Immuno. 7:33. Inone method, such transgenic animals are produced by incapacitating theendogenous loci encoding the heavy and light immunoglobulin chainstherein, and inserting loci encoding human heavy and light chainproteins into the genome thereof. Partially modified animals, that isthose having less than the full complement of modifications, are thencross-bred to obtain an animal having all of the desired immune systemmodifications. When administered an immunogen, these transgenic animalsproduce antibodies with human (rather than, e.g., murine) amino acidsequences, including variable regions which are immunospecific for theseantigens. See PCT App. Nos. PCT/US96/05928 and PCT/US93/06926.Additional methods are described in U.S. Pat. No. 5,545,807, PCT App.Nos. PCT/US91/245 and PCT/GB89/01207, and in European Patent Nos.546073B1 and 546073A1. Human antibodies can also be produced by theexpression of recombinant DNA in host cells or by expression inhybridoma cells as described herein.

In an alternative embodiment, human antibodies can also be produced fromphage-display libraries (Hoogenboom et al., 1991, J. Mol. Biol. 227:381;Marks et al., 1991, J. Mol. Biol. 222:581). These processes mimic immuneselection through the display of antibody repertoires on the surface offilamentous bacteriophage, and subsequent selection of phage by theirbinding to an antigen of choice. One such technique is described in PCTApp. No. PCT/US98/17364, which describes the isolation of high affinityand functional agonistic antibodies for MPL- and msk-receptors usingsuch an approach.

Chimeric, CDR grafted, and humanized antibodies are typically producedby recombinant methods. Nucleic acids encoding the antibodies areintroduced into host cells and expressed using materials and proceduresdescribed herein. In a preferred embodiment, the antibodies are producedin mammalian host cells, such as CHO cells. Monoclonal (e.g., human)antibodies may be produced by the expression of recombinant DNA in hostcells or by expression in hybridoma cells as described herein.

The anti-CHL antibodies of the invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays (Sola, MonoclonalAntibodies: A Manual of Techniques 147-158 (CRC Press, Inc., 1987)) forthe detection and quantitation of CHL polypeptides. The antibodies willbind CHL polypeptides with an affinity that is appropriate for the assaymethod being employed.

For diagnostic applications, in certain embodiments, anti-CHL antibodiesmay be labeled with a detectable moiety. The detectable moiety can beany one that is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²⁵I, ⁹⁹Tc, ¹¹¹In, or ⁶⁷Ga; afluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkalinephosphatase, □-galactosidase, or horseradish peroxidase (Bayer, et al.,1990, Meth. Enz. 184:138-63).

Competitive binding assays rely on the ability of a labeled standard(e.g., a CHL polypeptide, or an immunologically reactive portionthereof) to compete with the test sample analyte (an CHL polypeptide)for binding with a limited amount of anti-CHL antibody. The amount of aCHL polypeptide in the test sample is inversely proportional to theamount of standard that becomes bound to the antibodies. To facilitatedetermining the amount of standard that becomes bound, the antibodiestypically are insolubilized before or after the competition, so that thestandard and analyte that are bound to the antibodies may convenientlybe separated from the standard and analyte which remain unbound.

Sandwich assays typically involve the use of two antibodies, eachcapable of binding to a different immunogenic portion, or epitope, ofthe protein to be detected and/or quantitated. In a sandwich assay, thetest sample analyte is typically bound by a first antibody which isimmobilized on a solid support, and thereafter a second antibody bindsto the analyte, thus forming an insoluble three-part complex. See, e.g.,U.S. Pat. No. 4,376,110. The second antibody may itself be labeled witha detectable moiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assays). For example, one type of sandwich assay isan enzyme-linked immunosorbent assay (ELISA), in which case thedetectable moiety is an enzyme.

The selective binding agents, including anti-CHL antibodies, are alsouseful for in vivo imaging. An antibody labeled with a detectable moietymay be administered to an animal, preferably into the bloodstream, andthe presence and location of the labeled antibody in the host assayed.The antibody may be labeled with any moiety that is detectable in ananimal, whether by nuclear magnetic resonance, radiology, or otherdetection means known in the art.

Selective binding agents of the invention, including antibodies, may beused as therapeutics. These therapeutic agents are generally agonists orantagonists, in that they either enhance or reduce, respectively, atleast one of the biological activities of a CHL polypeptide. In oneembodiment, antagonist antibodies of the invention are antibodies orbinding fragments thereof which are capable of specifically binding to aCHL polypeptide and which are capable of inhibiting or eliminating thefunctional activity of a CHL polypeptide in vivo or in vitro. Inpreferred embodiments, the selective binding agent, e.g., an antagonistantibody, will inhibit the functional activity of a CHL polypeptide byat least about 50%, and preferably by at least about 80%. In anotherembodiment, the selective binding agent may be an anti-CHL polypeptideantibody that is capable of interacting with a CHL polypeptide bindingpartner (a ligand or receptor) thereby inhibiting or eliminating CHLpolypeptide activity in vitro or in vivo. Selective binding agents,including agonist and antagonist anti-CHL polypeptide antibodies, areidentified by screening assays that are well known in the art.

The invention also relates to a kit comprising CHL selective bindingagents (such as antibodies) and other reagents useful for detecting CHLpolypeptide levels in biological samples. Such reagents may include adetectable label, blocking serum, positive and negative control samples,and detection reagents.

Microarrays

It will be appreciated that DNA microarray technology can be utilized inaccordance with the present invention. DNA microarrays are miniature,high-density arrays of nucleic acids positioned on a solid support, suchas glass. Each cell or element within the array contains numerous copiesof a single nucleic acid species that acts as a target for hybridizationwith a complementary nucleic acid sequence (e.g., mRNA). In expressionprofiling using DNA microarray technology, mRNA is first extracted froma cell or tissue sample and then converted enzymatically tofluorescently labeled cDNA. This material is hybridized to themicroarray and unbound cDNA is removed by washing. The expression ofdiscrete genes represented on the array is then visualized byquantitating the amount of labeled cDNA that is specifically bound toeach target nucleic acid molecule. In this way, the expression ofthousands of genes can be quantitated in a high throughput, parallelmanner from a single sample of biological material.

This high throughput expression profiling has a broad range ofapplications with respect to the CHL molecules of the invention,including, but not limited to: the identification and validation of CHLdisease-related genes as targets for therapeutics; molecular toxicologyof related CHL molecules and inhibitors thereof; stratification ofpopulations and generation of surrogate markers for clinical trials; andenhancing related CHL polypeptide small molecule drug discovery byaiding in the identification of selective compounds in high throughputscreens.

Chemical Derivatives

Chemically modified derivatives of CHL polypeptides may be prepared byone skilled in the art, given the disclosures described herein. CHLpolypeptide derivatives are modified in a manner that isdifferent—either in the type or location of the molecules naturallyattached to the polypeptide. Derivatives may include molecules formed bythe deletion of one or more naturally-attached chemical groups. Thepolypeptide comprising the amino acid sequence of any of SEQ ID NO: 2,SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12, or other CHL polypeptide,may be modified by the covalent attachment of one or more polymers. Forexample, the polymer selected is typically water-soluble so that theprotein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. Included within thescope of suitable polymers is a mixture of polymers. Preferably, fortherapeutic use of the end- product preparation, the polymer will bepharmaceutically acceptable.

The polymers each may be of any molecular weight and may be branched orunbranched. The polymers each typically have an average molecular weightof between about 2 kDa to about 100 kDa (the term “about” indicatingthat in preparations of a water-soluble polymer, some molecules willweigh more, some less, than the stated molecular weight). The averagemolecular weight of each polymer is preferably between about 5 kDa andabout 50 kDa, more preferably between about 12 kDa and about 40 kDa andmost preferably between about 20 kDa and about 35 kDa.

Suitable water-soluble polymers or mixtures thereof include, but are notlimited to, N-linked or O-linked carbohydrates, sugars, phosphates,polyethylene glycol (PEG) (including the forms of PEG that have beenused to derivatize proteins, including mono-(C₁-C₁₀), alkoxy-, oraryloxy-polyethylene glycol), monomethoxy-polyethylene glycol, dextran(such as low molecular weight dextran of, for example, about 6 kD),cellulose, or other carbohydrate based polymers, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols(e.g., glycerol), and polyvinyl alcohol. Also encompassed by the presentinvention are bifunctional crosslinking molecules which may be used toprepare covalently attached CHL polypeptide multimers.

In general, chemical derivatization may be performed under any suitablecondition used to react a protein with an activated polymer molecule.Methods for preparing chemical derivatives of polypeptides willgenerally comprise the steps of: (a) reacting the polypeptide with theactivated polymer molecule (such as a reactive ester or aldehydederivative of the polymer molecule) under conditions whereby thepolypeptide comprising the amino acid sequence of any of SEQ ID NO: 2,SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12, or other CHL polypeptide,becomes attached to one or more polymer molecules, and (b) obtaining thereaction products. The optimal reaction conditions will be determinedbased on known parameters and the desired result. For example, thelarger the ratio of polymer molecules to protein, the greater thepercentage of attached polymer molecule. In one embodiment, the CHLpolypeptide derivative may have a single polymer molecule moiety at theamino-terminus. See, e.g., U.S. Pat. No. 5,234,784.

The pegylation of a polypeptide may be specifically carried out usingany of the pegylation reactions known in the art. Such reactions aredescribed, for example, in the following references: Francis et al.,1992, Focus on Growth Factors 3:4-10; European Patent Nos. 0154316 and0401384; and U.S. Pat. No. 4,179,337. For example, pegylation may becarried out via an acylation reaction or an alkylation reaction with areactive polyethylene glycol molecule (or an analogous reactivewater-soluble polymer) as described herein. For the acylation reactions,a selected polymer should have a single reactive ester group. Forreductive alkylation, a selected polymer should have a single reactivealdehyde group. A reactive aldehyde is, for example, polyethylene glycolpropionaldehyde, which is water stable, or mono C₁-C₁₀ alkoxy or aryloxyderivatives thereof (see U.S. Pat. No. 5,252,714).

In another embodiment, CHL polypeptides may be chemically coupled tobiotin. The biotin/CHL polypeptide molecules are then allowed to bind toavidin, resulting in tetravalent avidin/biotin/CHL polypeptidemolecules. CHL polypeptides may also be covalently coupled todinitrophenol (DNP) or trinitrophenol (TNP) and the resulting conjugatesprecipitated with anti-DNP or anti-TNP-IgM to form decameric conjugateswith a valency of 10.

Generally, conditions that may be alleviated or modulated by theadministration of the present CHL polypeptide derivatives include thosedescribed herein for CHL polypeptides. However, the CHL polypeptidederivatives disclosed herein may have additional activities, enhanced orreduced biological activity, or other characteristics, such as increasedor decreased half-life, as compared to the non-derivatized molecules.

Genetically Engineered Non-Human Animals

Additionally included within the scope of the present invention arenon-human animals such as mice, rats, or other rodents; rabbits, goats,sheep, or other farm animals, in which the genes encoding native CHLpolypeptide have been disrupted (i.e., “knocked out”) such that thelevel of expression of CHL polypeptide is significantly decreased orcompletely abolished. Such animals may be prepared using techniques andmethods such as those described in U.S. Pat. No. 5,557,032.

The present invention further includes non-human animals such as mice,rats, or other rodents; rabbits, goats, sheep, or other farm animals, inwhich either the native form of a CHL gene for that animal or aheterologous CHL gene is over-expressed by the animal, thereby creatinga “transgenic” animal. Such transgenic animals may be prepared usingwell known methods such as those described in U.S. Pat. No 5,489,743 andPCT Pub. No. WO 94/28122.

The present invention further includes non-human animals in which thepromoter for one or more of the CHL polypeptides of the presentinvention is either activated or inactivated (e.g., by using homologousrecombination methods) to alter the level of expression of one or moreof the native CHL polypeptides.

These non-human animals may be used for drug candidate screening. Insuch screening, the impact of a drug candidate on the animal may bemeasured. For example, drug candidates may decrease or increase theexpression of the CHL gene. In certain embodiments, the amount of CHLpolypeptide that is produced may be measured after the exposure of theanimal to the drug candidate. Additionally, in certain embodiments, onemay detect the actual impact of the drug candidate on the animal. Forexample, over-expression of a particular gene may result in, or beassociated with, a disease or pathological condition. In such cases, onemay test a drug candidate's ability to decrease expression of the geneor its ability to prevent or inhibit a pathological condition. In otherexamples, the production of a particular metabolic product such as afragment of a polypeptide, may result in, or be associated with, adisease or pathological condition. In such cases, one may test a drugcandidate's ability to decrease the production of such a metabolicproduct or its ability to prevent or inhibit a pathological condition.

Assaying for Other Modulators of CHL Polypeptide Activity

In some situations, it may be desirable to identify molecules that aremodulators, i.e., agonists or antagonists, of the activity of CHLpolypeptide. Natural or synthetic molecules that modulate CHLpolypeptide may be identified using one or more screening assays, suchas those described herein. Such molecules may be administered either inan ex vivo manner or in an in vivo manner by injection, or by oraldelivery, implantation device, or the like.

“Test molecule” refers to a molecule that is under evaluation for theability to modulate (i.e., increase or decrease) the activity of a CHLpolypeptide. Most commonly, a test molecule will interact directly witha CHL polypeptide. However, it is also contemplated that a test moleculemay also modulate CHL polypeptide activity indirectly, such as byaffecting CHL gene expression, or by binding to a CHL polypeptidebinding partner (e.g., receptor or ligand). In one embodiment, a testmolecule will bind to a CHL polypeptide with an affinity constant of atleast about 10⁻⁶ M, preferably about 10⁻⁸ M, more preferably about 10⁻⁹M, and even more preferably about 10⁻¹⁰ M.

Methods for identifying compounds that interact with CHL polypeptidesare encompassed by the present invention. In certain embodiments, a CHLpolypeptide is incubated with a test molecule under conditions thatpermit the interaction of the test molecule with a CHL polypeptide, andthe extent of the interaction is measured. The test molecule can bescreened in a substantially purified form or in a crude mixture.

In certain embodiments, a CHL polypeptide agonist or antagonist may be aprotein, peptide, carbohydrate, lipid, or small molecular weightmolecule that interacts with CHL polypeptide to regulate its activity.Molecules which regulate CHL polypeptide expression include nucleicacids which are complementary to nucleic acids encoding a CHLpolypeptide, or are complementary to nucleic acids sequences whichdirect or control the expression of CHL polypeptide, and which act asanti-sense regulators of expression.

Once a test molecule has been identified as interacting with a CHLpolypeptide, the molecule may be further evaluated for its ability toincrease or decrease CHL polypeptide activity. The measurement of theinteraction of a test molecule with CHL polypeptide may be carried outin several formats, including cell-based binding assays, membranebinding assays, solution-phase assays, and immunoassays. In general, atest molecule is incubated with a CHL polypeptide for a specified periodof time, and CHL polypeptide activity is determined by one or moreassays for measuring biological activity.

The interaction of test molecules with CHL polypeptides may also beassayed directly using polyclonal or monoclonal antibodies in animmunoassay. Alternatively, modified forms of CHL polypeptidescontaining epitope tags as described herein may be used in solution andimmunoassays.

In the event that CHL polypeptides display biological activity throughan interaction with a binding partner (e.g., a receptor or a ligand), avariety of in vitro assays may be used to measure the binding of a CHLpolypeptide to the corresponding binding partner (such as a selectivebinding agent, receptor, or ligand). These assays may be used to screentest molecules for their ability to increase or decrease the rate and/orthe extent of binding of a CHL polypeptide to its binding partner. Inone assay, a CHL polypeptide is immobilized in the wells of a microtiterplate. Radiolabeled CHL polypeptide binding partner (for example,iodinated CHL polypeptide binding partner) and a test molecule can thenbe added either one at a time (in either order) or simultaneously to thewells. After incubation, the wells can be washed and counted forradioactivity, using a scintillation counter, to determine the extent towhich the binding partner bound to the CHL polypeptide. Typically, amolecule will be tested over a range of concentrations, and a series ofcontrol wells lacking one or more elements of the test assays can beused for accuracy in the evaluation of the results. An alternative tothis method involves reversing the “positions” of the proteins, i.e.,immobilizing CHL polypeptide binding partner to the microtiter platewells, incubating with the test molecule and radiolabeled CHLpolypeptide, and determining the extent of CHL polypeptide binding. See,e.g., Current Protocols in Molecular Biology, chap. 18 (Ausubel et al.,eds., Green Publishers Inc. and Wiley and Sons 1995).

As an alternative to radiolabeling, a CHL polypeptide or its bindingpartner may be conjugated to biotin, and the presence of biotinylatedprotein can then be detected using streptavidin linked to an enzyme,such as horse radish peroxidase (HRP) or alkaline phosphatase (AP),which can be detected colorometrically, or by fluorescent tagging ofstreptavidin. An antibody directed to a CHL polypeptide or to a CHLpolypeptide binding partner, and which is conjugated to biotin, may alsobe used for purposes of detection following incubation of the complexwith enzyme-linked streptavidin linked to AP or HRP.

A CHL polypeptide or a CHL polypeptide binding partner can also beimmobilized by attachment to agarose beads, acrylic beads, or othertypes of such inert solid phase substrates. The substrate-proteincomplex can be placed in a solution containing the complementary proteinand the test compound. After incubation, the beads can be precipitatedby centrifugation, and the amount of binding between a CHL polypeptideand its binding partner can be assessed using the methods describedherein. Alternatively, the substrate-protein complex can be immobilizedin a column with the test molecule and complementary protein passingthrough the column. The formation of a complex between a CHL polypeptideand its binding partner can then be assessed using any of the techniquesdescribed herein (e.g., radiolabelling or antibody binding).

Another in vitro assay that is useful for identifying a test moleculewhich increases or decreases the formation of a complex between a CHLpolypeptide binding protein and a CHL polypeptide binding partner is asurface plasmon resonance detector system such as the BIAcore assaysystem (Pharmacia, Piscataway, N.J.). The BIAcore system is utilized asspecified by the manufacturer. This assay essentially involves thecovalent binding of either CHL polypeptide or a CHL polypeptide bindingpartner to a dextran-coated sensor chip that is located in a detector.The test compound and the other complementary protein can then beinjected, either simultaneously or sequentially, into the chambercontaining the sensor chip. The amount of complementary protein thatbinds can be assessed based on the change in molecular mass that isphysically associated with the dextran-coated side of the sensor chip,with the change in molecular mass being measured by the detector system.

In some cases, it may be desirable to evaluate two or more testcompounds together for their ability to increase or decrease theformation of a complex between a CHL polypeptide and a CHL polypeptidebinding partner. In these cases, the assays set forth herein can bereadily modified by adding such additional test compound(s) eithersimultaneously with, or subsequent to, the first test compound. Theremainder of the steps in the assay are as set forth herein.

In vitro assays such as those described herein may be usedadvantageously to screen large numbers of compounds for an effect on theformation of a complex between a CHL polypeptide and CHL polypeptidebinding partner. The assays may be automated to screen compoundsgenerated in phage display, synthetic peptide, and chemical synthesislibraries.

Compounds which increase or decrease the formation of a complex betweena CHL polypeptide and a CHL polypeptide binding partner may also bescreened in cell culture using cells and cell lines expressing eitherCHL polypeptide or CHL polypeptide binding partner. Cells and cell linesmay be obtained from any mammal, but preferably will be from human orother primate, canine, or rodent sources. The binding of a CHLpolypeptide to cells expressing CHL polypeptide binding partner at thesurface is evaluated in the presence or absence of test molecules, andthe extent of binding may be determined by, for example, flow cytometryusing a biotinylated antibody to a CHL polypeptide binding partner. Cellculture assays can be used advantageously to further evaluate compoundsthat score positive in protein binding assays described herein.

Cell cultures can also be used to screen the impact of a drug candidate.For example, drug candidates may decrease or increase the expression ofthe CHL gene. In certain embodiments, the amount of CHL polypeptide or aCHL polypeptide fragment that is produced may be measured after exposureof the cell culture to the drug candidate. In certain embodiments, onemay detect the actual impact of the drug candidate on the cell culture.For example, the over-expression of a particular gene may have aparticular impact on the cell culture. In such cases, one may test adrug candidate's ability to increase or decrease the expression of thegene or its ability to prevent or inhibit a particular impact on thecell culture. In other examples, the production of a particularmetabolic product such as a fragment of a polypeptide, may result in, orbe associated with, a disease or pathological condition. In such cases,one may test a drug candidate's ability to decrease the production ofsuch a metabolic product in a cell culture.

Internalizing Proteins

The tat protein sequence (from HIV) can be used to internalize proteinsinto a cell. See, e.g., Falwell et al., 1994, Proc. Natl. Acad. Sci.U.S.A. 91:664-68. For example, an 11 amino acid sequence(Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO: 18) of the HIV tat protein (termedthe “protein transduction domain,” or TAT PDT) has been described asmediating delivery across the cytoplasmic membrane and the nuclearmembrane of a cell. See Schwarze et al., 1999, Science 285:1569-72; andNagahara et al., 1998, Nat. Med. 4:1449-52. In these procedures,FITC-constructs (FITC-labeled G-G-G-G-Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO:19), which penetrate tissues following intraperitoneal administration,are prepared, and the binding of such constructs to cells is detected byfluorescence-activated cell sorting (FACS) analysis. Cells treated witha tat-□-gal fusion protein will demonstrate □-gal activity. Followinginjection, expression of such a construct can be detected in a number oftissues, including liver, kidney, lung, heart, and brain tissue. It isbelieved that such constructs undergo some degree of unfolding in orderto enter the cell, and as such, may require a refolding following entryinto the cell.

It will thus be appreciated that the tat protein sequence may be used tointernalize a desired polypeptide into a cell. For example, using thetat protein sequence, a CHL antagonist (such as an anti-CHL selectivebinding agent, small molecule, soluble receptor, or antisenseoligonucleotide) can be administered intracellularly to inhibit theactivity of a CHL molecule. As used herein, the term “CHL molecule”refers to both CHL nucleic acid molecules and CHL polypeptides asdefined herein. Where desired, the CHL protein itself may also beinternally administered to a cell using these procedures. See also,Straus, 1999, Science 285:1466-67.

Cell Source Identification Using CHL Polypeptide

In accordance with certain embodiments of the invention, it may beuseful to be able to determine the source of a certain cell typeassociated with a CHL polypeptide. For example, it may be useful todetermine the origin of a disease or pathological condition as an aid inselecting an appropriate therapy. In certain embodiments, nucleic acidsencoding a CHL polypeptide can be used as a probe to identify cellsdescribed herein by screening the nucleic acids of the cells with such aprobe. In other embodiments, one may use anti-CHL polypeptide antibodiesto test for the presence of CHL polypeptide in cells, and thus,determine if such cells are of the types described herein.

CHL Polypeptide Compositions and Administration

Therapeutic compositions are within the scope of the present invention.Such CHL polypeptide pharmaceutical compositions may comprise atherapeutically effective amount of a CHL polypeptide or a CHL nucleicacid molecule in admixture with a pharmaceutically or physiologicallyacceptable formulation agent selected for suitability with the mode ofadministration. Pharmaceutical compositions may comprise atherapeutically effective amount of one or more CHL polypeptideselective binding agents in admixture with a pharmaceutically orphysiologically acceptable formulation agent selected for suitabilitywith the mode of administration.

Acceptable formulation materials preferably are nontoxic to recipientsat the dosages and concentrations employed.

The pharmaceutical composition may contain formulation materials formodifying, maintaining, or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption, or penetration of thecomposition. Suitable formulation materials include, but are not limitedto, amino acids (such as glycine, glutamine, asparagine, arginine, orlysine), antimicrobials, antioxidants (such as ascorbic acid, sodiumsulfite, or sodium hydrogen-sulfite), buffers (such as borate,bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids),bulking agents (such as mannitol or glycine), chelating agents (such asethylenediamine tetraacetic acid (EDTA)), complexing agents (such ascaffeine, polyvinylpyrrolidone, beta-cyclodextrin, orhydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,disaccharides, and other carbohydrates (such as glucose, mannose, ordextrins), proteins (such as serum albumin, gelatin, orimmunoglobulins), coloring, flavoring and diluting agents, emulsifyingagents, hydrophilic polymers (such as polyvinylpyrrolidone), lowmolecular weight polypeptides, salt-forming counterions (such assodium), preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide),solvents (such as glycerin, propylene glycol, or polyethylene glycol),sugar alcohols (such as mannitol or sorbitol), suspending agents,surfactants or wetting agents (such as pluronics; PEG; sorbitan esters;polysorbates such as polysorbate 20 or polysorbate 80; triton;tromethamine; lecithin; cholesterol or tyloxapal), stability enhancingagents (such as sucrose or sorbitol), tonicity enhancing agents (such asalkali metal halides—preferably sodium or potassium chloride—or mannitolsorbitol), delivery vehicles, diluents, excipients and/or pharmaceuticaladjuvants. See Remington's Pharmaceutical Sciences (18th Ed., A. R.Gennaro, ed., Mack Publishing Company 1990.

The optimal pharmaceutical composition will be determined by a skilledartisan depending upon, for example, the intended route ofadministration, delivery format, and desired dosage. See, e.g.,Remington's Pharmaceutical Sciences, supra. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the CHL molecule.

The primary vehicle or carrier in a pharmaceutical composition may beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier for injection may be water, physiological saline solution, orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. Other exemplary pharmaceutical compositions comprise Trisbuffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, whichmay further include sorbitol or a suitable substitute. In one embodimentof the present invention, CHL polypeptide compositions may be preparedfor storage by mixing the selected composition having the desired degreeof purity with optional formulation agents (Remington's PharmaceuticalSciences, supra) in the form of a lyophilized cake or an aqueoussolution. Further, the CHL polypeptide product may be formulated as alyophilizate using appropriate excipients such as sucrose.

The CHL polypeptide pharmaceutical compositions can be selected forparenteral delivery. Alternatively, the compositions may be selected forinhalation or for delivery through the digestive tract, such as orally.The preparation of such pharmaceutically acceptable compositions iswithin the skill of the art.

The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at a slightly lowerpH, typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable, aqueous solution comprising thedesired CHL molecule in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which a CHL molecule is formulated as a sterile,isotonic solution, properly preserved. Yet another preparation caninvolve the formulation of the desired molecule with an agent, such asinjectable microspheres, bio-erodible particles, polymeric compounds(such as polylactic acid or polyglycolic acid), beads, or liposomes,that provides for the controlled or sustained release of the productwhich may then be delivered via a depot injection. Hyaluronic acid mayalso be used, and this may have the effect of promoting sustainedduration in the circulation. Other suitable means for the introductionof the desired molecule include implantable drug delivery devices.

In one embodiment, a pharmaceutical composition may be formulated forinhalation. For example, CHL polypeptide may be formulated as a drypowder for inhalation. CHL polypeptide or nucleic acid moleculeinhalation solutions may also be formulated with a propellant foraerosol delivery. In yet another embodiment, solutions may be nebulized.Pulmonary administration is further described in PCT Pub. No. WO94/20069, which describes the pulmonary delivery of chemically modifiedproteins.

It is also contemplated that certain formulations may be administeredorally. In one embodiment of the present invention, CHL polypeptidesthat are administered in this fashion can be formulated with or withoutthose carriers customarily used in the compounding of solid dosage formssuch as tablets and capsules. For example, a capsule may be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the CHL polypeptide. Diluents, flavorings,low melting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders may also be employed.

Another pharmaceutical composition may involve an effective quantity ofCHL polypeptides in a mixture with non-toxic excipients that aresuitable for the manufacture of tablets. By dissolving the tablets insterile water, or another appropriate vehicle, solutions can be preparedin unit-dose form. Suitable excipients include, but are not limited to,inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional CHL polypeptide pharmaceutical compositions will be evidentto those skilled in the art, including formulations involving CHLpolypeptides in sustained- or controlled-delivery formulations.Techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See, e.g., PCT/US93/00829, which describes thecontrolled release of porous polymeric microparticles for the deliveryof pharmaceutical compositions.

Additional examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices may includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 andEuropean Patent No. 058481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22:547-56),poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (European Patent No. 133988).Sustained-release compositions may also include liposomes, which can beprepared by any of several methods known in the art. See, e.g., Eppsteinet al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-92; and European PatentNos. 036676, 088046, and 143949.

The CHL pharmaceutical composition to be used for in vivo administrationtypically must be sterile. This may be accomplished by filtrationthrough sterile filtration membranes. Where the composition islyophilized, sterilization using this method may be conducted eitherprior to, or following, lyophilization and reconstitution. Thecomposition for parenteral administration may be stored in lyophilizedform or in a solution. In addition, parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations may bestored either in a ready-to-use form or in a form (e.g., lyophilized)requiring reconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

The effective amount of a CHL pharmaceutical composition to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which the CHLmolecule is being used, the route of administration, and the size (bodyweight, body surface, or organ size) and condition (the age and generalhealth) of the patient. Accordingly, the clinician may titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect. A typical dosage may range from about 0.1 □g/kg to up to about100 mg/kg or more, depending on the factors mentioned above. In otherembodiments, the dosage may range from 0.1 □g/kg up to about 100 mg/kg;or 1 □g/kg up to about 100 mg/kg; or 5 □g/kg up to about 100 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the CHL molecule in the formulation being used. Typically, aclinician will administer the composition until a dosage is reached thatachieves the desired effect. The composition may therefore beadministered as a single dose, as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages may be ascertained through use ofappropriate dose-response data.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g., orally; through injection byintravenous, intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intraocular, intraarterial,intraportal, or intralesional routes; by sustained release systems; orby implantation devices. Where desired, the compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device.

Alternatively or additionally, the composition may be administeredlocally via implantation of a membrane, sponge, or other appropriatematerial onto which the desired molecule has been absorbed orencapsulated. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed-release bolus, or continuousadministration.

In some cases, it may be desirable to use CHL polypeptide pharmaceuticalcompositions in an ex vivo manner. In such instances, cells, tissues, ororgans that have been removed from the patient are exposed to CHLpolypeptide pharmaceutical compositions after which the cells, tissues,or organs are subsequently implanted back into the patient.

In other cases, a CHL polypeptide can be delivered by implanting certaincells that have been genetically engineered, using methods such as thosedescribed herein, to express and secrete the CHL polypeptide. Such cellsmay be animal or human cells, and may be autologous, heterologous, orxenogeneic. Optionally, the cells may be immortalized. In order todecrease the chance of an immunological response, the cells may beencapsulated to avoid infiltration of surrounding tissues. Theencapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

As discussed herein, it may be desirable to treat isolated cellpopulations (such as stem cells, lymphocytes, red blood cells,chondrocytes, neurons, and the like) with one or more CHL polypeptides.This can be accomplished by exposing the isolated cells to thepolypeptide directly, where it is in a form that is permeable to thecell membrane.

Additional embodiments of the present invention relate to cells andmethods (e.g., homologous recombination and/or other recombinantproduction methods) for both the in vitro production of therapeuticpolypeptides and for the production and delivery of therapeuticpolypeptides by gene therapy or cell therapy. Homologous and otherrecombination methods may be used to modify a cell that contains anormally transcriptionally-silent CHL gene, or an under-expressed gene,and thereby produce a cell which expresses therapeutically efficaciousamounts of CHL polypeptides.

Homologous recombination is a technique originally developed fortargeting genes to induce or correct mutations in transcriptionallyactive genes. Kucherlapati, 1989, Prog. in Nucl. Acid Res. & Mol. Biol.36:301. The basic technique was developed as a method for introducingspecific mutations into specific regions of the mammalian genome (Thomaset al., 1986, Cell 44:419-28; Thomas and Capecchi, 1987, Cell 51:503-12;Doetschman et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:8583-87) or tocorrect specific mutations within defective genes (Doetschman et al.,1987, Nature 330:576-78). Exemplary homologous recombination techniquesare described in U.S. Pat. No. 5,272,071; European Patent Nos. 9193051and 505500; PCT/US90/07642, and PCT Pub No. WO 91/09955).

Through homologous recombination, the DNA sequence to be inserted intothe genome can be directed to a specific region of the gene of interestby attaching it to targeting DNA. The targeting DNA is a nucleotidesequence that is complementary (homologous) to a region of the genomicDNA. Small pieces of targeting DNA that are complementary to a specificregion of the genome are put in contact with the parental strand duringthe DNA replication process. It is a general property of DNA that hasbeen inserted into a cell to hybridize, and therefore, recombine withother pieces of endogenous DNA through shared homologous regions. Ifthis complementary strand is attached to an oligonucleotide thatcontains a mutation or a different sequence or an additional nucleotide,it too is incorporated into the newly synthesized strand as a result ofthe recombination. As a result of the proofreading function, it ispossible for the new sequence of DNA to serve as the template. Thus, thetransferred DNA is incorporated into the genome.

Attached to these pieces of targeting DNA are regions of DNA that mayinteract with or control the expression of a CHL polypeptide, e.g.,flanking sequences. For example, a promoter/enhancer element, asuppressor, or an exogenous transcription modulatory element is insertedin the genome of the intended host cell in proximity and orientationsufficient to influence the transcription of DNA encoding the desiredCHL polypeptide. The control element controls a portion of the DNApresent in the host cell genome. Thus, the expression of the desired CHLpolypeptide may be achieved not by transfection of DNA that encodes theCHL gene itself, but rather by the use of targeting DNA (containingregions of homology with the endogenous gene of interest) coupled withDNA regulatory segments that provide the endogenous gene sequence withrecognizable signals for transcription of a CHL gene.

In an exemplary method, the expression of a desired targeted gene in acell (i.e., a desired endogenous cellular gene) is altered viahomologous recombination into the cellular genome at a preselected site,by the introduction of DNA which includes at least a regulatorysequence, an exon, and a splice donor site. These components areintroduced into the chromosomal (genomic) DNA in such a manner thatthis, in effect, results in the production of a new transcription unit(in which the regulatory sequence, the exon, and the splice donor sitepresent in the DNA construct are operatively linked to the endogenousgene). As a result of the introduction of these components into thechromosomal DNA, the expression of the desired endogenous gene isaltered.

Altered gene expression, as described herein, encompasses activating (orcausing to be expressed) a gene which is normally silent (unexpressed)in the cell as obtained, as well as increasing the expression of a genewhich is not expressed at physiologically significant levels in the cellas obtained. The embodiments further encompass changing the pattern ofregulation or induction such that it is different from the pattern ofregulation or induction that occurs in the cell as obtained, andreducing (including eliminating) the expression of a gene which isexpressed in the cell as obtained.

One method by which homologous recombination can be used to increase, orcause, CHL polypeptide production from a cell's endogenous CHL geneinvolves first using homologous recombination to place a recombinationsequence from a site-specific recombination system (e.g., Cre/loxP,FLP/FRT) (Sauer, 1994, Curr. Opin. Biotechnol., 5:521-27; Sauer, 1993,Methods Enzymol., 225:890-900) upstream of (i.e., 5′ to) the cell'sendogenous genomic CHL polypeptide coding region. A plasmid containing arecombination site homologous to the site that was placed just upstreamof the genomic CHL polypeptide coding region is introduced into themodified cell line along with the appropriate recombinase enzyme. Thisrecombinase causes the plasmid to integrate, via the plasmid'srecombination site, into the recombination site located just upstream ofthe genomic CHL polypeptide coding region in the cell line (Baubonis andSauer, 1993, Nucleic Acids Res. 21:2025-29; O'Gorman et al., 1991,Science 251:1351-55). Any flanking sequences known to increasetranscription (e.g., enhancer/promoter, intron, translational enhancer),if properly positioned in this plasmid, would integrate in such a manneras to create a new or modified transcriptional unit resulting in de novoor increased CHL polypeptide production from the cell's endogenous CHLgene.

A further method to use the cell line in which the site specificrecombination sequence had been placed just upstream of the cell'sendogenous genomic CHL polypeptide coding region is to use homologousrecombination to introduce a second recombination site elsewhere in thecell line's genome. The appropriate recombinase enzyme is thenintroduced into the two-recombination-site cell line, causing arecombination event (deletion, inversion, and translocation) (Sauer,1994, Curr. Opin. Biotechnol., 5:521-27; Sauer, 1993, Methods Enzymol.,225:890-900) that would create a new or modified transcriptional unitresulting in de novo or increased CHL polypeptide production from thecell's endogenous CHL gene.

An additional approach for increasing, or causing, the expression of CHLpolypeptide from a cell's endogenous CHL gene involves increasing, orcausing, the expression of a gene or genes (e.g., transcription factors)and/or decreasing the expression of a gene or genes (e.g.,transcriptional repressors) in a manner which results in de novo orincreased CHL polypeptide production from the cell's endogenous CHLgene. This method includes the introduction of a non-naturally occurringpolypeptide (e.g., a polypeptide comprising a site specific DNA bindingdomain fused to a transcriptional factor domain) into the cell such thatde novo or increased CHL polypeptide production from the cell'sendogenous CHL gene results.

The present invention further relates to DNA constructs useful in themethod of altering expression of a target gene. In certain embodiments,the exemplary DNA constructs comprise: (a) one or more targetingsequences, (b) a regulatory sequence, (c) an exon, and (d) an unpairedsplice-donor site. The targeting sequence in the DNA construct directsthe integration of elements (a)-(d) into a target gene in a cell suchthat the elements (b)-(d) are operatively linked to sequences of theendogenous target gene. In another embodiment, the DNA constructscomprise: (a) one or more targeting sequences, (b) a regulatorysequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) asplice-acceptor site, wherein the targeting sequence directs theintegration of elements (a)-(f) such that the elements of (b)-(f) areoperatively linked to the endogenous gene. The targeting sequence ishomologous to the preselected site in the cellular chromosomal DNA withwhich homologous recombination is to occur. In the construct, the exonis generally 3′ of the regulatory sequence and the splice-donor site is3′ of the exon.

If the sequence of a particular gene is known, such as the nucleic acidsequence of CHL polypeptide presented herein, a piece of DNA that iscomplementary to a selected region of the gene can be synthesized orotherwise obtained, such as by appropriate restriction of the native DNAat specific recognition sites bounding the region of interest. Thispiece serves as a targeting sequence upon insertion into the cell andwill hybridize to its homologous region within the genome. If thishybridization occurs during DNA replication, this piece of DNA, and anyadditional sequence attached thereto, will act as an Okazaki fragmentand will be incorporated into the newly synthesized daughter strand ofDNA. The present invention, therefore, includes nucleotides encoding aCHL polypeptide, which nucleotides may be used as targeting sequences.

CHL polypeptide cell therapy, e.g., the implantation of cells producingCHL polypeptides, is also contemplated. This embodiment involvesimplanting cells capable of synthesizing and secreting a biologicallyactive form of CHL polypeptide. Such CHL polypeptide-producing cells canbe cells that are natural producers of CHL polypeptides or may berecombinant cells whose ability to produce CHL polypeptides has beenaugmented by transformation with a gene encoding the desired CHLpolypeptide or with a gene augmenting the expression of CHL polypeptide.Such a modification may be accomplished by means of a vector suitablefor delivering the gene as well as promoting its expression andsecretion. In order to minimize a potential immunological reaction inpatients being administered a CHL polypeptide, as may occur with theadministration of a polypeptide of a foreign species, it is preferredthat the natural cells producing CHL polypeptide be of human origin andproduce human CHL polypeptide. Likewise, it is preferred that therecombinant cells producing CHL polypeptide be transformed with anexpression vector containing a gene encoding a human CHL polypeptide.

Implanted cells may be encapsulated to avoid the infiltration ofsurrounding tissue. Human or non-human animal cells may be implanted inpatients in biocompatible, semipermeable polymeric enclosures ormembranes that allow the release of CHL polypeptide, but that preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissue. Alternatively, thepatient's own cells, transformed to produce CHL polypeptides ex vivo,may be implanted directly into the patient without such encapsulation.

Techniques for the encapsulation of living cells are known in the art,and the preparation of the encapsulated cells and their implantation inpatients may be routinely accomplished. For example, Baetge et al. (PCTPub. No. WO 95/05452 and PCT/US94/09299) describe membrane capsulescontaining genetically engineered cells for the effective delivery ofbiologically active molecules. The capsules are biocompatible and areeasily retrievable. The capsules encapsulate cells transfected withrecombinant DNA molecules comprising DNA sequences coding forbiologically active molecules operatively linked to promoters that arenot subject to down-regulation in vivo upon implantation into amammalian host. The devices provide for the delivery of the moleculesfrom living cells to specific sites within a recipient. In addition, seeU.S. Pat. Nos. 4,892,538; 5,011,472; and 5,106,627. A system forencapsulating living cells is described in PCT Pub. No. WO 91/10425(Aebischer et al.). See also, PCT Pub. No. WO 91/10470 (Aebischer etal.); Winn et al., 1991, Exper. Neurol. 113:322-29; Aebischer et al.,1991, Exper. Neurol. 111:269-75; and Tresco et al., 1992, ASAIO38:17-23.

In vivo and in vitro gene therapy delivery of CHL polypeptides is alsoenvisioned. One example of a gene therapy technique is to use the CHLgene (either genomic DNA, cDNA, and/or synthetic DNA) encoding a CHLpolypeptide which may be operably linked to a constitutive or induciblepromoter to form a “gene therapy DNA construct.” The promoter may behomologous or heterologous to the endogenous CHL gene, provided that itis active in the cell or tissue type into which the construct will beinserted. Other components of the gene therapy DNA construct mayoptionally include DNA molecules designed for site-specific integration(e.g., endogenous sequences useful for homologous recombination),tissue-specific promoters, enhancers or silencers, DNA molecules capableof providing a selective advantage over the parent cell, DNA moleculesuseful as labels to identify transformed cells, negative selectionsystems, cell specific binding agents (as, for example, for celltargeting), cell-specific internalization factors, transcription factorsenhancing expression from a vector, and factors enabling vectorproduction.

A gene therapy DNA construct can then be introduced into cells (eitherex vivo or in vivo) using viral or non-viral vectors. One means forintroducing the gene therapy DNA construct is by means of viral vectorsas described herein. Certain vectors, such as retroviral vectors, willdeliver the DNA construct to the chromosomal DNA of the cells, and thegene can integrate into the chromosomal DNA. Other vectors will functionas episomes, and the gene therapy DNA construct will remain in thecytoplasm.

In yet other embodiments, regulatory elements can be included for thecontrolled expression of the CHL gene in the target cell. Such elementsare turned on in response to an appropriate effector. In this way, atherapeutic polypeptide can be expressed when desired. One conventionalcontrol means involves the use of small molecule dimerizers or rapalogsto dimerize chimeric proteins which contain a small molecule-bindingdomain and a domain capable of initiating a biological process, such asa DNA-binding protein or transcriptional activation protein (see PCTPub. Nos. WO 96/41865, WO 97/31898, and WO 97/31899). The dimerizationof the proteins can be used to initiate transcription of the transgene.

An alternative regulation technology uses a method of storing proteinsexpressed from the gene of interest inside the cell as an aggregate orcluster. The gene of interest is expressed as a fusion protein thatincludes a conditional aggregation domain that results in the retentionof the aggregated protein in the endoplasmic reticulum. The storedproteins are stable and inactive inside the cell. The proteins can bereleased, however, by administering a drug (e.g., small molecule ligand)that removes the conditional aggregation domain and thereby specificallybreaks apart the aggregates or clusters so that the proteins may besecreted from the cell. See Aridor et al., 2000, Science 287:816-17 andRivera et al., 2000, Science 287:826-30.

Other suitable control means or gene switches include, but are notlimited to, the systems described herein. Mifepristone (RU486) is usedas a progesterone antagonist. The binding of a modified progesteronereceptor ligand-binding domain to the progesterone antagonist activatestranscription by forming a dimer of two transcription factors that thenpass into the nucleus to bind DNA. The ligand-binding domain is modifiedto eliminate the ability of the receptor to bind to the natural ligand.The modified steroid hormone receptor system is further described inU.S. Pat. No. 5,364,791 and PCT Pub. Nos. WO 96/40911 and WO 97/10337.

Yet another control system uses ecdysone (a fruit fly steroid hormone)which binds to and activates an ecdysone receptor (cytoplasmicreceptor). The receptor then translocates to the nucleus to bind aspecific DNA response element (promoter from ecdysone-responsive gene).The ecdysone receptor includes a transactivation domain, DNA-bindingdomain, and ligand-binding domain to initiate transcription. Theecdysone system is further described in U.S. Pat. No. 5,514,578 and PCTPub. Nos. WO 97/38117, WO 96/37609, and WO 93/03162.

Another control means uses a positive tetracycline-controllabletransactivator. This system involves a mutated tet repressor proteinDNA-binding domain (mutated tet R-4 amino acid changes which resulted ina reverse tetracycline-regulated. transactivator protein, i.e., it bindsto a tet operator in the presence of tetracycline) linked to apolypeptide which activates transcription. Such systems are described inU.S. Pat. Nos. 5,464,758, 5,650,298, and 5,654,168.

Additional expression control systems and nucleic acid constructs aredescribed in U.S. Pat. Nos. 5,741,679 and 5,834,186, to InnovirLaboratories Inc.

In vivo gene therapy may be accomplished by introducing the geneencoding CHL polypeptide into cells via local injection of a CHL nucleicacid molecule or by other appropriate viral or non-viral deliveryvectors. Hefti 1994, Neurobiology 25:1418-35. For example, a nucleicacid molecule encoding a CHL polypeptide may be contained in anadeno-associated virus (AAV) vector for delivery to the targeted cells(see, e.g., Johnson, PCT Pub. No. WO 95/34670; PCT App. No.PCT/US95/07178). The recombinant AAV genome typically contains AAVinverted terminal repeats flanking a DNA sequence encoding a CHLpolypeptide operably linked to functional promoter and polyadenylationsequences.

Alternative suitable viral vectors include, but are not limited to,retrovirus, adenovirus, herpes simplex virus, lentivirus, hepatitisvirus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus,rhabdovirus, paramyxovirus, and papilloma virus vectors. U.S. Pat. No.5,672,344 describes an in vivo viral-mediated gene transfer systeminvolving a recombinant neurotrophic HSV-1 vector. U.S. Pat. No.5,399,346 provides examples of a process for providing a patient with atherapeutic protein by the delivery of human cells which have beentreated in vitro to insert a DNA segment encoding a therapeutic protein.Additional methods and materials for the practice of gene therapytechniques are described in U.S. Pat. No. 5,631,236 (involvingadenoviral vectors), U.S. Pat. No. 5,672,510 (involving retroviralvectors), U.S. Pat. No. 5,635,399 (involving retroviral vectorsexpressing cytokines).

Nonviral delivery methods include, but are not limited to,liposome-mediated transfer, naked DNA delivery (direct injection),receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation, and microparticle bombardment (e.g.,gene gun). Gene therapy materials and methods may also include induciblepromoters, tissue-specific enhancer-promoters, DNA sequences designedfor site-specific integration, DNA sequences capable of providing aselective advantage over the parent cell, labels to identify transformedcells, negative selection systems and expression control systems (safetymeasures), cell-specific binding agents (for cell targeting),cell-specific internalization factors, and transcription factors toenhance expression by a vector as well as methods of vector manufacture.Such additional methods and materials for the practice of gene therapytechniques are described in U.S. Pat. No. 4,970,154 (involvingelectroporation techniques), U.S. Pat. No. 5,679,559 (describing alipoprotein-containing system for gene delivery), U.S. Pat. No.5,676,954 (involving liposome carriers), U.S. Pat. No. 5,593,875(describing methods for calcium phosphate transfection), and U.S. Pat.No. 4,945,050 (describing a process wherein biologically activeparticles are propelled at cells at a speed whereby the particlespenetrate the surface of the cells and become incorporated into theinterior of the cells), and PCT Pub. No. WO 96/40958 (involving nuclearligands).

It is also contemplated that CHL gene therapy or cell therapy canfurther include the delivery of one or more additional polypeptide(s) inthe same or a different cell(s). Such cells may be separately introducedinto the patient, or the cells may be contained in a single implantabledevice, such as the encapsulating membrane described above, or the cellsmay be separately modified by means of viral vectors.

A means to increase endogenous CHL polypeptide expression in a cell viagene therapy is to insert one or more enhancer elements into the CHLpolypeptide promoter, where the enhancer elements can serve to increasetranscriptional activity of the CHL gene. The enhancer elements usedwill be selected based on the tissue in which one desires to activatethe gene—enhancer elements known to confer promoter activation in thattissue will be selected. For example, if a gene encoding a CHLpolypeptide is to be “turned on” in T-cells, the lck promoter enhancerelement may be used. Here, the functional portion of the transcriptionalelement to be added may be inserted into a fragment of DNA containingthe CHL polypeptide promoter (and optionally, inserted into a vectorand/or 5′ and/or 3′ flanking sequences) using standard cloningtechniques. This construct, known as a “homologous recombinationconstruct,” can then be introduced into the desired cells either ex vivoor in vivo.

Gene therapy also can be used to decrease CHL polypeptide expression bymodifying the nucleotide sequence of the endogenous promoter. Suchmodification is typically accomplished via homologous recombinationmethods. For example, a DNA molecule containing all or a portion of thepromoter of the CHL gene selected for inactivation can be engineered toremove and/or replace pieces of the promoter that regulatetranscription. For example, the TATA box and/or the binding site of atranscriptional activator of the promoter may be deleted using standardmolecular biology techniques; such deletion can inhibit promoteractivity thereby repressing the transcription of the corresponding CHLgene. The deletion of the TATA box or the transcription activatorbinding site in the promoter may be accomplished by generating a DNAconstruct comprising all or the relevant portion of the CHL polypeptidepromoter (from the same or a related species as the CHL gene to beregulated) in which one or more of the TATA box and/or transcriptionalactivator binding site nucleotides are mutated via substitution,deletion and/or insertion of one or more nucleotides. As a result, theTATA box and/or activator binding site has decreased activity or isrendered completely inactive. This construct, which also will typicallycontain at least about 500 bases of DNA that correspond to the native(endogenous) 5′ and 3′ DNA sequences adjacent to the promoter segmentthat has been modified, may be introduced into the appropriate cells(either ex vivo or in vivo) either directly or via a viral vector asdescribed herein. Typically, the integration of the construct into thegenomic DNA of the cells will be via homologous recombination, where the5′ and 3′ DNA sequences in the promoter construct can serve to helpintegrate the modified promoter region via hybridization to theendogenous chromosomal DNA.

Therapeutic Uses

CHL nucleic acid molecules, polypeptides, and agonists and antagoniststhereof can be used to treat, diagnose, ameliorate, or prevent a numberof diseases, disorders, or conditions, including those recited herein.

CHL polypeptide agonists and antagonists include those molecules whichregulate CHL polypeptide activity and either increase or decrease atleast one activity of the mature form of the CHL polypeptide. Agonistsor antagonists may be co-factors, such as a protein, peptide,carbohydrate, lipid, or small molecular weight molecule, which interactwith CHL polypeptide and thereby regulate its activity. Potentialpolypeptide agonists or antagonists include antibodies that react witheither soluble or membrane-bound forms of CHL polypeptides that comprisepart or all of the extracellular domains of the said proteins. Moleculesthat regulate CHL polypeptide expression typically include nucleic acidsencoding CHL polypeptide that can act as anti-sense regulators ofexpression.

Preliminary biological and biochemical characterization suggests severaltherapeutic utilities for CHL polypeptides. CHL polypeptides, fragments,variants, and/or derivatives may be used to prevent or treat bonediseases such as osteopetrosis and osteoporosis, aid in tissueregeneration and wound healing, or function in hematopoietic stemcell-genesis and expansion.

In clinical settings, the adult body is the major therapeutic target,and proteins that are produced and work in a normal healthy body may,therefore, give significant therapeutic benefits when they have beenshown to function in organ homeostasis. In an adult organism, one of themajor roles of the BMP-family of gene products—specifically BMP2 andBMP4—is the regulation of bone-mass. Since BMP 1 has been shown tocleave and inactivate CHD and probably CHL polypeptide, and has beenisolated with BMP2 and BMP3 from bone (Wozney et al., 1988, Science242:1528-34; Celeste et al, 1990, Proc. Nat. Acad. Sci. USA 87:9843-47), it is possible that CHL polypeptide and CHD play keyregulatory roles in osteogenesis. This implies that one of therate-limiting steps for the control of bone mass may be the regulationof the BMP2/4 activity through CHL polypeptide and BMP 1, as well asthrough Noggin. Thus, by administering CHL polypeptides or anti-CHLantibodies, the amount and activity of CHL polypeptide may be controlledand the bone density of in an adult decreased or increased as desired.

The direct delivery of BMP4 or other BMP-family members to theregenerating bone through the blood stream appears to be astraightforward therapeutic concept for treatment of osteopetrosis.However, it may be difficult to accomplish since BMP4 is known to travelonly a short distance in vivo (Jones et al., 1996, Curr. Biol.6:1468-75). By forming a complex with CHL polypeptide, BMP may migratefurther—as in the case of CHD in an embryo leading to the formation of aconcentration gradient for BMP4 (Jones and Smith, 1998, Dev. Biol.194:12-17). Patients suffering from either osteopetrosis or osteoporosiswould benefit from improved treatments.

BMP polypeptides have been shown to function in organ formation duringlate embryogenesis. It is well-known that organ formation in embryonickidney, lung, and gut are affected by BMP4 expression (Hogan 1996, GenesDev. 10:1580-94). CHL potypeptide is shown herein to be expressed in thelung and small intestine, while CHD is known to be expressed in thekidney. Thus, it is possible that by using a combination of BMP4 and CHLpolypeptide (or of BMP and CHD) the proliferation and differentiation ofprogenitor cells in these tissues could be controlled permitting for theregulation of tissue regeneration or wound healing in vivo.

Agonists or antagonists of CHL polypeptide function may be used(simultaneously or sequentially) in combination with one or morecytokines, growth factors, antibiotics, anti-inflammatories, and/orchemotherapeutic agents as is appropriate for the condition beingtreated.

Other diseases or disorders caused by or mediated by undesirable levelsof CHL polypeptides are encompassed within the scope of the invention.Undesirable levels include excessive levels of CHL polypeptides andsub-normal levels of CHL polypeptides.

Uses of CHL Nucleic Acids and Polypeptides

Nucleic acid molecules of the invention (including those that do notthemselves encode biologically active polypeptides) may be used to mapthe locations of the CHL gene and related genes on chromosomes. Mappingmay be done by techniques known in the art, such as PCR amplificationand in situ hybridization.

CHL nucleic acid molecules (including those that do not themselvesencode biologically active polypeptides), may be useful as hybridizationprobes in diagnostic assays to test, either qualitatively orquantitatively, for the presence of a CHL nucleic acid molecule inmammalian tissue or bodily fluid samples.

Other methods may also be employed where it is desirable to inhibit theactivity of one or more CHL polypeptides. Such inhibition may beeffected by nucleic acid molecules that are complementary to andhybridize to expression control sequences (triple helix formation) or toCHL mRNA. For example, antisense DNA or RNA molecules, which have asequence that is complementary to at least a portion of a CHL gene canbe introduced into the cell. Anti-sense probes may be designed byavailable techniques using the sequence of the CHL gene disclosedherein. Typically, each such antisense molecule will be complementary tothe start site (5′ end) of each selected CHL gene. When the antisensemolecule then hybridizes to the corresponding CHL mRNA, translation ofthis mRNA is prevented or reduced. Anti-sense inhibitors provideinformation relating to the decrease or absence of a CHL polypeptide ina cell or organism.

Alternatively, gene therapy may be employed to create adominant-negative inhibitor of one or more CHL polypeptides. In thissituation, the DNA encoding a mutant polypeptide of each selected CHLpolypeptide can be prepared and introduced into the cells of a patientusing either viral or non-viral methods as described herein. Each suchmutant is typically designed to compete with endogenous polypeptide inits biological role.

In addition, a CHL polypeptide, whether biologically active or not, maybe used as an immunogen, that is, the polypeptide contains at least oneepitope to which antibodies may be raised. Selective binding agents thatbind to a CHL polypeptide (as described herein) may be used for in vivoand in vitro diagnostic purposes, including, but not limited to, use inlabeled form to detect the presence of CHL polypeptide in a body fluidor cell sample. The antibodies may also be used to prevent, treat, ordiagnose a number of diseases and disorders, including those recitedherein. The antibodies may bind to a CHL polypeptide so as to diminishor block at least one activity characteristic of a CHL polypeptide, ormay bind to a polypeptide to increase at least one activitycharacteristic of a CHL polypeptide (including by increasing thepharmacokinetics of the CHL polypeptide).

The CHL polypeptides of the present invention can be used to clone CHLpolypeptide receptors, using an expression cloning strategy.Radiolabeled (¹²⁵Iodine) CHL polypeptide or affinity/activity-tagged CHLpolypeptide (such as an Fc fusion or an alkaline phosphatase fusion) canbe used in binding assays to identify a cell type or cell line or tissuethat expresses CHL polypeptide receptors. RNA isolated from such cellsor tissues can be converted to cDNA, cloned into a mammalian expressionvector, and transfected into mammalian cells (such as COS or 293 cells)to create an expression library. A radiolabeled or tagged CHLpolypeptide can then be used as an affinity ligand to identify andisolate from this library the subset of cells that express the CHLpolypeptide receptors on their surface. DNA can then be isolated fromthese cells and transfected into mammalian cells to create a secondaryexpression library in which the fraction of cells expressing CHLpolypeptide receptors is many-fold higher than in the original library.This enrichment process can be repeated iteratively until a singlerecombinant clone containing a CHL polypeptide receptor is isolated.Isolation of the CHL polypeptide receptors is useful for identifying ordeveloping novel agonists and antagonists of the CHL polypeptidesignaling pathway. Such agonists and antagonists include soluble CHLpolypeptide receptors, anti-CHL polypeptide receptor antibodies, smallmolecules, or antisense oligonucleotides, and they may be used fortreating, preventing, or diagnosing one or more of the diseases ordisorders described herein.

In addition to indicating that CHL polypeptides interact with andinhibit the activity of BMP4, preliminary biological and biochemicalcharacterization suggests several other utilities for CHL polypeptides.

Since BMP2 is the most closely related BMP-family member to BMP4, CHLpolypeptide may also inhibit the function of BMP2. In addition,interactions with other BMP-family members may also be detected.Furthermore, CHL polypeptide may also interact with a novel set ofproteins that are not related to the BMP family. The murine, rat, andhuman CHL nucleic acids described herein are useful tools for obtainingthe corresponding recombinant proteins. The recombinant CHL polypeptidesof the present invention can be used to identify proteins that interactwith this protein. For example, the CHL polypeptides may also be used todetermine whether CHL polypeptide interacts directly with a membrane orintracellular receptor.

The murine, rat, and human CHL nucleic acids of the present inventionare also useful tools for isolating the corresponding chromosomal CHLpolypeptide genes. For example, mouse chromosomal DNA containing CHLsequences can be used to construct knockout mice, thereby permitting anexamination of the in vivo role for CHL polypeptide. The human CHLgenomic DNA can be used to identify heritable tissue-degeneratingdiseases.

As described herein, CHL mRNA has been identified in a set of bonemarrow stroma cell lines that are known to support hematopoietic stemcells and early progenitor cells, but has not been identified in stromacell lines which support only committed progenitor cells (see Example4). On the other hand, the CHL mRNA is detected in bone marrow, but notin fetal liver or in peripheral blood leukocytes. These observationsimply that CHL polypeptide or CHL-interacting proteins might have somefunction in regulating hematopoietic stem cell survival and maintenance,specifically in the bone marrow environment. The CHL polypeptides andCHL nucleic acids described herein may provide useful tools for in vitroexpansion of hematopoietic stem cells. Alternatively, BMP4 or otherputative CHL-interacting molecules can be used for regulating thesurvival and maintenance of hematopoietic stem cells.

BMP4 is an essential factor for generating hematopoietic progenitorcells from the mouse ES cells. However, the effective concentration ofBMP4 falls into a narrow range (0.5 ng/ml to 5 ng/ml), which isconsistent with the idea that the difference in the active concentrationof BMP correlates with the difference in the resulting cell-type fromthe totipotent epiblast. A system for the reproducible in vitrogeneration of hematopoietic stem cells from ES cells has not yet beendisclosed. However, it may be achieved by precise control of theconcentration of BMP4. The CHL polypeptides, anti-CHL antibodies, andCHL nucleic acids of the present invention would be useful tools foroptimizing the culturing conditions for the in vitro generation ofhematopoietic stem cells from ES cells.

Primitive hematopoietic stem cells have been recently defined in themouse yolk sac (Yoder et al., 1997, Proc. Natl. Acad. Sci. USA94:6776-80). This subclass of hematopoietic stem cells does not exhibita marrow-repopulating activity in adults. However, when exposed to anewborn liver environment, the primitive stem cells are converted tolong-term marrow-repopulating stem cells (i.e., definitive stem cells).This can also be accomplished by culturing the primitive stem cells oncertain stroma cell lines. The long-term maintenance of the definitivestem cells in culture and the long-term generation of the definitivestem cells from the primitive stem cells in culture have not yet beendistinguished. The preferential expression of CHL mRNA in the stromacell lines which support the definitive stem cell survival andmaintenance suggests that interactions between BMP and CHL polypeptideor between BMP and CHD system might also function in the stem cellmaintenance arid/or generation. Thus the recombinant CHL polypeptidesand CHL antibodies of the present invention may be useful tools for theboth long-term maintenance and generation of the definitivehematopoietic stem cells in vitro. Alternatively, BMP4 or putative,novel CHL-interacting molecules can be used for controlling theseprocesses.

The primitive hematopoietic stem cells have yet to be fullycharacterized. While primitive stem cells may be of a lymphocytic celltype, such cells may also be mesodermal precursors which are able togenerate hematopoietic cell types as well as other mesodermal progeny.In support of this idea, adult bone marrow has recently been shown tocontain endothelial progenitor cells, cells that regenerate liver(Petersen et al., 1999, Science 284:1168-70), and a common stem cellthat has a capability of deriving endothelial cells, muscle cells andhematopoietic cells in vivo (Ferrari et al., 1998, Science 279:1528-30).Furthermore, the osteoblast cell lineage, which consists of the bonemarrow stroma, is known to be derived from a mesenchymal stem cell thatis present in bone marrow. The possibility that a common mesodermal stemcell is responsible for the generation of both stroma and hematopoieticcells has also been previously speculated.

Nevertheless, the fact that growth and differentiation of osteoblastsare regulated by BMP and that CHL polypeptide is expressed in bonemarrow suggest that the BMP/CHL system might be involved in biologicalprocesses in which the bone marrow-mesodermal stem cells are involved.In this respect, the recombinant CHL polypeptides and CHL antibodies ofthe present invention may be useful for characterization of suchmulti-lineage stem cells.

As described herein, primitive hematopoietic stem cells have been foundin the mouse yolk sac. This class of hematopoietic stem cells has beenshown to possess marrow-repopulating activity only followingpre-exposure in a new-born liver environment. Since the presence of aprimitive stem cell activity has never been investigated in theconventional hematopoietic sites of bone marrow, umbilical cord blood,and fetal liver, this suggests that there may be as yet unidentified(i.e. primitive) hematopoietic stem cells in these tissues. If theprimitive hematopoietic stem cell is present in such hematopoietictissues, it can be a novel target for ex vivo expansion, and could be abetter target for gene transfer since such cells are more primitive thandefinitive stem cells. The concept that the conversion of primitive stemcells to definitive stem cells can also be achieved in culture oncertain stroma cell lines makes this idea clinically feasible.

As discussed herein, through the use of BMPiCHD or BMP/CHL it may bepossible to regulate primitive hematopoietic stem cells and therebycontrol adult-marrow repopulating stem cells. Alternatively, it may bepossible to control stem cell genesis from a mesodermal stem cell. Thus,the CHL polypeptides and nucleic acids of the present invention, alongwith BMP, may be useful for ex vivo expansion of hematopoietic stemcells and gene therapy performed through such cells.

Deposits of cDNA encoding murine, rat, and human CHL polypeptide andhuman CHLd5 polypeptide, subcloned into pSPORT1 (Gibco BRL), havingAccession Nos. PTA-961, PTA-962, PTA-963, or PTA-964, were made with theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209 on Nov. 16, 1999.

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

EXAMPLE 1 Cloning of the Murine CHL Polypeptide Gene

Generally, materials and methods as described in Sambrook et al. suprawere used to clone and analyze the gene encoding murine CHL polypeptide.

Sequences encoding murine CHL polypeptide were isolated from anormalized cDNA library of the mouse bone marrow stroma cell line, OP9,as a sequence with statistically meaningful homology to the CR domainsof chicken CHD. The normalized cDNA library was prepared by using thepolymerase chain reaction (PCR) based normalization procedure ofTakahashi and Ko, 1994, Genomics 23: 202-10.

Part of the normalized OP9 library of 3×10⁵ independent clones wasscreened for clones containing signal-like sequences using theamylase-based yeast signal trap method (U.S. patent applcation Ser. No.09/026,959). Individual colonies that gave a positive signal werepicked, and the cDNA insert was amplified from each yeast clone by PCRusing vector primers. The partial clone carrying the 5′-half of thecoding region (clone tmsn1-00001-h4) was detected among approximately400 inserts sequenced.

A full-length cDNA clone (clone tmsn1-00001-h4-wze/agp-61975-a1) wasthen isolated from an OP9 full-length cDNA library constructed in thepcDNA3.1 expression vector (Invitrogen). Forty-eight individualsub-pools of the full-length cDNA library, each containing 2×10⁴independent clones, were screened using a probe generated from thepartial CHL nucleic acid sequence by PCR using the primers 2125-05(5′-A-G-T-G-C-C-C-A-G-C-T-T-T-A-G-T-C-C-A-C-3′; SEQ ID NO: 20) and2125-06 (5′-G-A-G-A-T-G-A-G-G-A-A-T-A-T-G-C-A-C-G-G-3′; SEQ ID NO: 21).The resulting 350 bp PCR fragment contained 267 bp of the CHLpolypeptide coding region and 84 bp 5′ non-coding sequence. Fourpositive clones were obtained from a screen of 2×10⁴ recombinant cDNAclones. The clone with the longest insert size (clonetmsnl-00001-h4-wze/agp-61975-a1) was sequenced.

Sequence analysis of the full-length cDNA for murine CHL polypeptide(clone 1-2) indicated that the gene comprises a 999 bp open readingframe encoding a protein of 333 amino acids (FIGS. 1A-1C). The murineCHL polypeptide sequence is predicted to contain a signal peptide (FIG.1A, predicted signal peptide indicated by underline).

EXAMPLE 2 Cloning of the Rat CHL Polypeptide Gene

Generally, materials and methods as described in Sambrook et al. suprawere used to clone and analyze the gene encoding rat CHL polypeptide.

Sequences encoding rat CHL polypeptide were isolated from a normalizedrat prostate cDNA library as a result of expression-sequence-tag (EST)sequencing. A partial rat CHL nucleic acid sequence (clonesrpb2-00279-a4) of 370 base pairs in length and encoding a polypeptidefragment that is 39% identical to CHD was identified through suchanalysis. The corresponding full-length rat CHL cDNA (clonesrpb2-00279-a4-wz) was subsequently isolated by screening anoligo(dT)-primed rat prostate cDNA library using the partial rat CHLnucleic acid sequence as a probe.

Sequence analysis of the full-length cDNA for rat CHL polypeptideindicated that the gene comprises a 1146 bp open reading frame encodinga protein of 382 amino acids (FIGS. 2A-2D). The rat CHL polypeptidesequence is predicted to contain a signal peptide (FIG. 2A, predictedsignal peptide indicated by underline).

EXAMPLE 3 Cloning of the Human CHL Polypeptide Gene

Generally, materials and methods as described in Sambrook et al. suprawere used to clone and analyze the gene encoding human CHL polypeptide.

Sequences encoding human CHL polypeptide were isolated from a humanfetal brain cDNA library (Stratagene) using the partial rat CHL cDNAclone as a probe. The human CHL cDNA clone that was recovered lacked theamino-terminal end of the coding sequence, and the 5′-end part of theCHL mRNA was cloned separately using RACE methodology using a humanheart marathon-ready cDNA kit (Clontech). The first round of PCR wasperformed using the 5′-primer supplied with the kit (AP1) and the genespecific primer 2127-58 (5′-G-A-C-A-T-C-T-G-A-C-T-C-G-G-C-T-G-C-3′; SEQID NO: 22). The second PCR amplification was performed using the5′-primer supplied with the kit (AP2) and the gene specific primer2212-48 (5′-T-C-A-C-G-C-A-G-T-A-A-A-C-C-A-A-C-3′; SEQ ID NO: 23).

The resulting PCR fragment was subcloned into the TOPO cloning vector(Invitrogen), the nucleotide sequence confirmed by sequencing, and the5′ human CHL fragment was then inserted into the partial human CHL cDNAdescribed above in order to reconstruct the full-length human CHL cDNA(termed srpb2-00279-a4-wze).

Sequence analysis of the full-length cDNA for human CHL polypeptideindicated that the gene comprises a 1356 bp open reading frame encodinga protein of 452 amino acids (FIGS. 3A-3C). The human CHL polypeptidesequence is predicted to contain a signal peptide (FIG. 3A, predictedsignal peptide indicated by underlined and/or double-underline).

A second form of human CHL cDNA, designated as CHLd5, was identifiedduring the course of constructing the human CHL-FLAG fusion polypeptide(see Example 6). A full-length human CHLd5 DNA fragment in which thestop codon was replaced by a Bam HI site was obtained by PCR using heartcDNA (Clonetech) as a template and the primers 2235-53(5′-C-G-G-A-A-T-T-C-G-C-C-A-C-C-A-T-G-G-G-A-G-G-C-A-T-G-A-A-A-T-A-C-A-T-C-T-T-T-3′;SEQ ID NO: 24) and 2235-54(5′-C-G-C-G-G-A-T-C-C-A-C-A-G-T-G-G-C-C-C-T-T-T-T-C-A-G-A-T-C-T-C-T-C-3′;SEQ ID NO: 25). The amplified PCR product was digested with Eco RI andBam HI, gel purified, and then inserted into the pFLAG-CMV5a expressionvector (Sigma). The resulting CHL-FLAG expression plasmid is designatedas pFLAGhCHLd5. This form of human CHL polypeptide has an internaldeletion of 5 amino acids (Gly³¹⁹-Lys³²⁰-Lys³²¹-Ala³²²-Lys³²³)immediately following CR3. Interestingly, the identical peptide ispresent in the murine CHL polypeptide, but is lacking in the rat CHLpolypeptide. Therefore, the rat CHL polypeptide may correspond to CHLd5.

Sequence analysis of the full-length cDNA for human CHLd5 polypeptideindicated that the gene comprises a 1341 bp open reading frame encodinga protein of 447 amino acids (FIGS. 4A-4C). The human CHLd5 polypeptidesequence is predicted to contain a signal peptide (FIG. 4A, predictedsignal peptide indicated by underlined and/or double-underline).

Computer analysis of the isolated murine, rat, and human sequencesindicated that they possessed three repeated CR motifs, in contrast tothe four repeats observed in CHD. Although CR motifs have been found ina number of other known proteins, including pro-collagen, a homologysearch in GenBank revealed that the CHL polypeptide motifs were mostclosely related to the CR motifs of CHD/SOG. The CR1 and CR3 of CHLpolypeptide were particularly found to be highly homologous to the CR3of CHD/SOD (indicated by gray boxes; FIG. 5). The structural arrangementof the three CR motifs in CHL polypeptide seems to correspond to that ofCR2-CR4 in CHD since CHL polypeptide lacks one CR motif as compared toCHD and also lacks the long gap between the CR1 and CR2 motifs of CHD.FIGS. 6A-6E illustrate the amino acid sequence alignment of human CHLpolypeptide, human CHLd5 polypeptide, murine CHL polypeptide, rat CHLpolypeptide, murine CHD, rat CHD, and human CHD. The murine CHLpolypeptide is notable in that it is approximately one-third the size ofthe 948 amino acid murine CHD protein.

EXAMPLE 4 CHL mRNA Expression

Multiple human or murine tissue northern blots (for mouse, mouse embryo,human, human II, and human immune system II, obtained from Clontech)were probed using a ³²P-dCTP-labeled 350 bp PCR fragment generated byamplification of the murine CHL cDNA clone (see Example 1).

The Northern blots were prehybridized in Express Hybridization solution(Clontech) for 1 hour at 68° C. and then were hybridized in the samesolution with the addition of 1.3 ng/mL of labeled probe for 1 hour at68° C. Following hybridization, the filters were washed three times in2×SSC and 0.05% SDS for 10 minutes at room temperature, and then twicein 0.1×SSC and 0.1% SDS for 20 minutes at 50° C. Following washing, theblots were subjected to autoradiography.

Among the adult murine tissues analyzed, transcripts of 4 kb in lengthhaving the highest expression were detected in the heart, brain, lung,and testis, transcripts with a moderate level of expression weredetected in skeletal muscle, and transcripts with a lower level ofexpression were detected in the spleen, liver, and kidney (FIG. 7A).Analysis of human tissue northern blots identified an abundant humantranscript of 4-7 kb in length that cross reacts with the murine probein the heart, brain, lung, placenta, prostate, small intestine and colon(mucosal lining). Lower levels of expression of this transcript weredetected in liver, skeletal muscle, kidney, pancreas, thymus, lymphnode, spleen, bone marrow, testis, and ovary (FIG. 8). Expression belowthe level of detection on northern blots was found in peripheral bloodleukocytes and fetal liver.

A comparative Northern analysis in mouse embryos provides additionalsupport for a relationship between CHL polypeptide and CHD/SOG. WhileCHD/SOG has been shown to be expressed in E7 embryos—and at much lowerlevels in E11, E15 and E17 embryos (Pappano et al., 1998, Genomics52:236-39)—CHL RNA, in contrast, was found to be expressed in E11, E15,and E17 embryos but not in E7 embryos (FIG. 7B). Furthermore, while CHLexpression is detected in the adult heart and lung expression of CHD/SOGin these tissues is very weak (Pappano et al., 1998 Genomics 52:236-39).In contrast, CHD/SOG expression is detected in the spleen, liver, andkidney (Pappano et al., 1998, Genomics 52:236-39), while CHL expressionin these tissues is very weak or below the level of detection. Theexpression pattern of CHL mRNA seems to contrast that of CHD/SOG withonly a few exceptions (i.e., brain and testis). Nevertheless, theseresults suggest that CHL polypeptide is not only structurally related toCHD/SOG but may also be functionally related.

Northern analyses were also performed on several bone-marrow-derivedstroma cell lines (FIGS. 9A-9B). CHL polypeptide seems to be expresseddifferentially according to their properties. The stroma cell lines F10and F4, which are known to support early hematopoietic progenitor cells(probably stem cells) in the formation of delayed cobblestone areas(CAs), were found to express CHL mRNA (FIG. 9A). However, the stromacell line D3—a variant supporting only mature hematopoietic progenitorcells in the formation of short-term CAs, does not express CHL mRNA. Inthis respect, CHL expression correlates with the ability of stroma cellsto support survival, maintenance, and differentiation of hematopoieticstem cells. Since bone marrow, but not fetal liver, was found to expressCHL mRNA (FIG. 8), it would be expected that CHL polypeptide isexpressed by a stroma cell population specific to bone marrow ratherthan to fetal liver. The results of the Northern analysis also suggestthat CHL polypeptide is probably not expressed in mature hematopoieticcell lineages, as peripheral blood leukocytes were found to not expressappreciable levels of CHL mRNA (FIG. 8).

The expression of CHL mRNA was localized by in situ hybridization. Insitu hybridization was performed as described in Harlow and Lane, UsingAntibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press,1999), using antisense-RNAs synthesized with SP6 RNA polymerase (Ambion,Austin, Tex.) from pSPmCHL5′. The pSPmCHL5′ vector was prepared byremoving the region between the Cla I and Not I sites of pSPORTmCHL andthen linearizing the vector with Eco RI.

The expression of CHL mRNA was analyzed in those tissues determined tobe CHL-expressing in the Northern blot analysis described above.Populations of fibroblast/connective tissue cells in the femalereproductive tract, the gastrointestinal tract (FIG. 10), and the outermedulla of the kidney (FIG. 11) were found to express CHL mRNA. In thebrain, CHL transcripts were localized in the cerebral cortex, theexternal plexiform layer of the olfactory bulb, and Purkinje cells ofthe cerebellum (FIG. 12). The weak expression of CHL mRNA detected inthe lung and liver by Northern analysis, was actually localized to bloodvessels supplying these tissues (FIG. 10). Weak expression of CHL mRNAdetected in the spleen and lymph nodes was localized to anon-lymphocytic cell population. Interestingly, in situ hybridizationrevealed additional sites of CHL mRNA expression. These additional sitesinclude skin mesenchyme and white adipose tissue. These observationssuggest that, in the adult mouse, CHL mRNA is expressed mainly in cellsof mesenchymal origin (with the exception of neuronal cells in thebrain).

In day 12.5 mouse embryos, a low level of signal was detected overcephalic mesenchyme in areas where the basioccipital and exoccipitalbones will form, over mesenchymal cells in the lower jaw, and adjacentto the dorsal root ganglia in areas where vertebrae will form. A verylow level of signal was also detected over the stomach wall (but not theepithelial lining of the stomach). In day 13.5 embryos, a signal wasdetected over the cartilage of the ribs, vertebra, limb bone (FIG. 13),and trachea, and possibly over mesenchymal condensations destined tobecome tendon. In day 14.5 through 18.5 embryos, a detectable signalover the cartilage of the developing bones (FIG. 13) was still present.In bones forming by endochondral formation, the signal appeared to beprimarily over the hypertrophic chondrocytes in the growth regions,while the signal was more diffuse and less restricted in, for example,the bones of the head, which develop by intramembranous formation (FIG.12). Also at these stages, the pattern of expression in thegastrointestinal tract was similar to that of the adult, with a lowlevel of signal being detected in a layer of fibroblast or smooth musclecells between the mucosa and mucularis. One difference between embryonicand adult CHL mRNA expression concerned the esophagus where a signal wasdetected in the embryo but not the adult. This difference may have beendue to the level of the embryonic sections that were examined.

At several embryonic stages, a signal was detected over relativelyundifferentiated mesenchymal/connective tissue-type cells. For example,in day 14.5 embryos, CHL mRNA expression was detected in, and adjacentto, the salivary glands. In addition, CHL mRNA expression was detectedin day 18.5 embryos in the subcutaneous tissue.

In summary, the strongest signal detected with the CHL mRNA probe wasdetected in the developing skeleton of the mouse embryo. Such expressionwas detected in the cartilage of bones undergoing either intramembranousor endochondral development. In the adult mouse CHL mRNA expression inbone or cartilage (as examined in the growth plate region of long bones,the articular cartilage of the joints, and tracheal cartilage) waseither not present or was below the level of sensitivity by in situanalysis. These observations support the argument that CHL mRNA may bean important regulator of bone and cartilage formation.

EXAMPLE 5 Chromosomal Mapping of the Murine CHL Polypeptide Gene

Fluorescence in situ hybridization (FISH) analsis was used to determinethe chromosomal localization of the murine CHL gene (Shi et al., 1997,Genomics 45:42-47). A FISH probe was prepared from a BAC clone isolatedfrom the Mouse ES-129/SvJ II BAC chromosome DNA library (Genome Systems)by PCR using standard techniques and primers corresponding to the secondCR domain of the murine CHL gene(5′-T-T-A-C-C-A-C-C-A-G-T-G-A-A-C-A-A-T-A-A-G-G -3′; SEQ ID NO: 26 and5′-C-T-T-G-A-G-A-C-C-A-C-A-G-T-A-T-A-C-A-T-T-C-C-3′; SEQ ID NO: 27). Theisolated BAC clone, F1038, was further examined by PCR using standardtechniques and primers covering the 5′ untranslated region and signalpeptide region of the murine CHL gene(5′-A-G-T-G-C-C-C-A-G-C-T-T-T-A-G-T-C-C-A-C-3′; SEQ ID NO: 28 and5′-G-T-T-C-T-G-T-T-T-T-G-C-T-T-C-C-T-T-C-T-A-G-3′; SEQ ID NO: 29). Fromthis analysis, it was concluded that the F1038 clone contains at leastthose exons of the murine CHL gene spanning from the 5′ untranslatedregion to the second CR domain. The murine CHL gene was mapped usingF1038 DNA as a probe. It was found to be on the X chromosome.

A partial sequence of the human CHL gene, derived from the q22.1-23region of the human X chromosome, was found in GenBank (Accession no.AL049176). In order to localize the murine CHL gene on the X chromosome,the X centromere-specific P1 clone (#6856) was also used as aco-hybridization probe (Shi et al., 1997, Genomics 45:42-47). A total of80 metaphase cells were analyzed with 72 exhibiting specific labeling.Of these, 10 were used for co-hybridization experiments. The murine CHLgene was located at a position, which was 89% of the distance from theheterochromatic-euchromatic boundary to the telomere of the Xchromosome, an area corresponding to band XF3. Therefore, it has beenconcluded that both human and murine CHL genes are located on a similarregion of the X chromosome.

EXAMPLE 6 Production of CHL Polypeptides

A. Expression of CHL Polypeptides in Bacteria

PCR is used to amplify template DNA sequences encoding a CHL polypeptideusing primers corresponding to the 5′ and 3′ ends of the sequence. Theamplified DNA products may be modified to contain restriction enzymesites to allow for insertion into expression vectors. PCR products aregel purified and inserted into expression vectors using standardrecombinant DNA methodology. An exemplary vector, such as pAMG21 (ATCCno. 98113) containing the lux promoter and a gene encoding kanamycinresistance is digested with Bam HI and Nde I for directional cloning ofinserted DNA. The ligated mixture is transformed into an E. coli hoststrain by electroporation and transformants are selected for kanamycinresistance. Plasmid DNA from selected colonies is isolated and subjectedto DNA sequencing to confirm the presence of the insert.

Transformed host cells are incubated in 2×YT medium containing 30 □g/mLkanamycin at 30° C. prior to induction. Gene expression is induced bythe addition of N-(3-oxohexanoyl)-dl-homoserine lactone to a finalconcentration of 30 ng/mL followed by incubation at either 30° C. or 37°C. for six hours. The expression of CHL polypeptide is evaluated bycentrifugation of the culture, resuspension and lysis of the bacterialpellets, and analysis of host cell proteins by SDS-polyacrylamide gelelectrophoresis.

Inclusion bodies containing CHL polypeptide are purified as follows.Bacterial cells are pelleted by centrifugation and resuspended in water.The cell suspension is lysed by sonication and pelleted bycentrifugation at 195,000×g for 5 to 10 minutes. The supernatant isdiscarded, and the pellet is washed and transferred to a homogenizer.The pellet is homogenized in 5 mL of a Percoll solution (75% liquidPercoll and 0.15 M NaCl) until uniformly suspended and then diluted andcentrifuged at 21,600×g for 30 minutes. Gradient fractions containingthe inclusion bodies are recovered and pooled. The isolated inclusionbodies are analyzed by SDS-PAGE.

A single band on an SDS polyacrylamide gel corresponding to E.coli-produced CHL polypeptide is excised from the gel, and theN-terminal amino acid sequence is determined essentially as described byMatsudaira et al., 1987, J. Biol. Chem. 262:10-35.

B. Construction of CHL Polypeptide Mammalian Expression Vectors

A FLAG-tagged murine CHL polypeptide expression construct was preparedas follows. A full-length murine CHL DNA fragment, in which the stopcodon was replaced by a BamHI site, was obtained by PCR using thefull-length murine cDNA clone as a template and the primers 2149-76(5′-G-C-T-A-G-C-G-G-C-C-G-C-G-C-C-A-C-C-A-T-G-G-A-T-G-G-C-A-T-G-A-A-A-T-A-C-A-T-C-A-T-T-T-C-3′;SEQ ID NO: 30) and 2149-77(5′-G-G-T-A-C-C-G-G-A-T-C-C-A-C-C-A-A-A-G-G-C-A-G-G-G-C-C-T-C-C-A-G-C-3′;SEQ ID NO: 31).

The amplified PCR product was digested with Not I and Bam HI, gelpurified, and then inserted into the pFLAG-CMV-5a expression vector(Sigma) with the FLAG-sequence attached in-frame with the CHL sequenceat its carboxyl-terminus. The fusion site between the CHL and FLAGsequences was subsequently confirmed by sequencing. The resultingCHL-FLAG expression plasmid is designated as pFLAGmCHL.

A FLAG-tagged murine CHD construct was prepared as follows. Murine CHDcDNA was isolated by RT-PCR from a mouse E7 embryo cDNA library(Clontech) using the primers 2170-06(5′-G-C-T-A-G-C-G-G-C-C-G-C-G-C-C-A-C-C-A-T-G-C-C-G-A-G-C-C-T-C-C-C-G-G-C-C-C-C-G-3′;SEQ ID NO: 32) and 2170-07(5′-G-G-A-T-C-C-G-T-C-G-A-C-G-G-A-G-T-G-C-T-C-C-G-C-T-T-C-T-T-T-C-T-C-C-A-G-3′;SEQ ID NO: 33).

The amplified PCR product was digested with Not I and Sal I, gelpurified, and then inserted into the pFLAG-CMV-5a expression vector.Eleven clones were isolated and the corresponding cDNA insertssequenced. To generate the final CHD-FLAG expression construct (labeledpFLAGmCHD), a 1.9 kb Eco RI—Bgl II fragment of one of the identifiedclones (clone #16) was replaced with the corresponding 1.9 kb fragmentof a separate clone (clone #3) to remove errors in the sequenceintroduced as a result of PCR amplification.

To analyze the expression of either CHL-FLAG or CHD-FLAG, the pFLAGmCHLand pFLAGmCHD expression constructs were first introduced into human293T cells using SuperFect transfection reagent (Qiagen), according tothe manufacturer's suggested protocols. Prior to, and immediatelyfollowing transfection, the 293T cells were maintained in DMEMsupplemented with 10% Fetal Calf Serum (FCS, Hyclone). Followingtransfection, the cells were incubated overnight at 37° C. and 5% CO₂,and then the culture medium was renewed with serum-free IMDM,supplemented with 5-15% Knockout SR (Gibco BRL), and the cells wereincubated for an additional 48 hours. The conditioned medium was thenremoved and CHL-FLAG and CHD-FLAG protein expression was analyzed byWestern blot analysis using the anti-FLAG antibody M2 (Sigma).Approximately, 1 □g/mL of the CHD-FLAG and CHL-FLAG proteins wereobtained.

To generate clones capable of stably expressing CHL-FLAG or CHD-FLAG,293 cells were transfected using the calcium phosphate method witheither linearized pFLAGmCHL or pFLAGmCHD and pGKneo (in a ratio of 30 to1). After incubating the cells for 36 to 48 hours at 37° C., thetransfected cell were renewed with fresh medium containing 800 □g/mLG418 (Gibco BRL) for 12 days, and 12 clones were isolated for eachtransfection reaction. The expression levels of the CHL-FLAG andCHD-FLAG proteins were determined by Western blot analysis using theanti-FLAG M2 antibody. The clones with the highest expression levels(about 0.1 □g of CHL-FLAG or CHD-FLAG/mL of cell supernatant) wereselected and expanded (FIG. 14A).

CHL-FLAG and CHD-FLAG proteins were partially purified from the selectedclones as follows. Clones expressing the proteins were expanded bygrowing the cells as suspension cells in a spinner culture with 293 SFM(Gibco BRL). Conditioned medium was collected and cell debris removed bycentrifugation. CHL-FLAG and CHD-FLAG proteins were isolated by affinitychromatography using anti-FLAG M2 affinity gel (Sigma) packed inpoly-prep chromatography columns (Bio-Rad). The bound fraction,containing the FLAG-tagged CHL polypeptide or CHD protein was eluted byadding 100 □g/mL FLAG peptide (Sigma).

The human CHL-FLAG protein was also constructed in a similar way. Afull-length human CHL DNA fragment in which the stop codon was replacedby a Bam HI site was obtained by PCR using the full-length human CHLcDNA clone as a template and the primers 2235-53 and 2235-54. Theamplified PCR product was digested with Eco RI and Bam HI, gel purified,and then inserted into the pFLAG-CMV5a expression vector (Sigma). Theresulting CHL-FLAG expression plasmid is designated as pFLAGhCHL. ThepFLAGhCHL plasmid was introduced into human 293T cells as described,conditioned medium was removed, and the human CHL-FLAG expression wasanalyzed by Western blot analysis with anti-FLAG antibody M2 (Sigma)(FIG. 14C).

C. Expression and Purification of CHL Polypeptide in Mammalian Cells

CHL polypeptide expression constructs are introduced into 293 EBNA orCHO cells using either a lipofection or calcium phosphate protocol.

To conduct functional studies on the CHL polypeptides that are produced,large quantities of conditioned media are generated from a pool ofhygromycin selected 293 EBNA clones. The cells are cultured in 500 cmNunc Triple Flasks to 80% confluence before switching to serum freemedia a week prior to harvesting the media. Conditioned media isharvested and frozen at −20° C. until purification.

Conditioned media is purified by affinity chromatography as describedbelow. The media is thawed and then passed through a 0.2 □m filter. AProtein G column is equilibrated with PBS at pH 7.0, and then loadedwith the filtered media. The column is washed with PBS until theabsorbance at A₂₈₀ reaches a baseline. CHL polypeptide is eluted fromthe column with 0.1 M Glycine-HCl at pH 2.7 and immediately neutralizedwith 1 M Tris-HCl at pH 8.5. Fractions containing CHL polypeptide arepooled, dialyzed in PBS, and stored at −70° C.

For Factor Xa cleavage of the human CHL polypeptide-Fc fusionpolypeptide, affinity chromatography-purified protein is dialyzed in 50mM Tris-HCl, 100 mM NaCl, 2 mM CaCl₂ at pH 8.0. The restriction proteaseFactor Xa is added to the dialyzed protein at 1/100 (w/w) and the sampleis digested overnight at room temperature.

EXAMPLE 7 The N-terminal Amino Acid Sequence Determination of the MatureCHL-FLAG and CHD-FLAG Proteins

The affinity purified murine CHL-FLAG and murine CHD-FLAG proteins wereseparated from contaminated proteins by the SDS-polyacrylamide gelelectrophoresis, and the major band was excised for amino acid sequencedetermination with the gas-phase peptide sequencer, Procise 494 (AppliedBiosystems). The results have indicated that the murine CHL-FLAGpolypeptide precursor (SEQ ID NO: 34) is cleaved between Thr²² andGlu²³, and that the murine CHD-FLAG protein precursor (SEQ ID NO: 36) iscleaved between Gly²⁶ and Thr²⁷ to generate the mature forms of murineCHL-FLAG polypeptide (SEQ ID NO: 35) and CHD-FLAG protein (SEQ ID NO:37).

EXAMPLE 8 Production of Anti-CHL Polypeptide Antibodies

Antibodies to CHL polypeptides may be obtained by immunization withpurified protein or with CHL peptides produced by biological or chemicalsynthesis. Suitable procedures for generating antibodies include thosedescribed in Hudson and Bay, Practical Immunology (2nd ed., BlackwellScientific Publications).

In one procedure for the production of antibodies, animals (typicallymice or rabbits) are injected with a CHL antigen (such as a CHLpolypeptide), and those with sufficient serum titer levels as determinedby ELISA are selected for hybridoma production. Spleens of immunizedanimals are collected and prepared as single cell suspensions from whichsplenocytes are recovered. The splenocytes are fused to mouse myelomacells (such as Sp2/0-Ag14 cells), are first incubated in DMEM with 200U/mL penicillin, 200 □g/mL streptomycin sulfate, and 4 mM glutamine, andare then incubated in HAT selection medium (hypoxanthine, aminopterin,and thymidine). After selection, the tissue culture supernatants aretaken from each fusion well and tested for anti-CHL antibody productionby ELISA.

Alternative procedures for obtaining anti-CHL antibodies may also beemployed, such as the immunization of transgenic mice harboring human Igloci for production of human antibodies, and the screening of syntheticantibody libraries, such as those generated by mutagenesis of anantibody variable domain.

EXAMPLE 9 Biological Activity of Murine CHL in Xenopus Embryos

To assay the biological effect of native murine CHL polypeptide inXenopus embryos, a vector carrying the full-length murine CHL cDNA(pcDNA3mCHL), was used for in vitro RNA synthesis. The vector was firstlinearized with Not I and the capped mRNA was then transcribed with T7RNA polymerase, using the MMESSAGE mMACHINE T7 kit (Ambion). FormCHL-FLAG, the Eco RI-Sca I fragment of FLAG-tagged CHL polypeptide wasfirst cloned into the Eco RI-Not I site of RN3 (Lemaire et al., 1995,Cell 81:85-94), and then linearized with Sfi I. Then, capped mRNA wastranscribed with T3 RNA polymerase. Following either synthesis,recovered RNA was subjected to two rounds of ethanol precipitation with0.5 M ammonium acetate to remove unincorporated nucleotides, and thenquantified by spectrophotometry at 260 nm.

Xenopus embryos were dejellied in 3% cysteine and staged as described inNieuwkoop and Faber, Normal Table of Xenopus laevis (Daudin, ed.,Garland Publishing, 1994). Embryos were placed into Steinberg's solutioncontaining 5% Ficoll and 5 nL of RNA was injected into the two ventralblastomeres of 4-cell stage embryos. Following injection with variousamounts of RNA, embryos were cultured in 10% Steinberg's solution for 48hours, and the embryos scored for ectopic axis. Of the embryos injectedwith 100 pg of either murine CHL-FLAG or native murine CHL RNA, allshowed hyperdorsalization phenotypes, in which the anterior portions ofthe embryos are enlarged and the posterior parts are missing. Thisresult is often observed when an excess amount of RNA carrying axisduplication activity has been injected into the embryo. The injection of30 pg RNA/embryo was found to be the optimal amount for generating anaxis duplication as a result of the introduction of the CHL polypeptideconstructs (FIGS. 15A-15B). An axis duplication rate for CHL polypeptideof 86.7% (26/30 embryos) was observed whereas the rate for uninjectedcontrol embryos was 0% (0/31 embryos). The axis duplication rate forCHL-FLAG with poly-A was 78.9% (15/19 embryos), which is similar to thatof the native CHL polypeptide without the FLAG-peptide and poly-A. As apositive control, experiments were also performed using the clonedmurine CHD-FLAG (lacking poly-A). An axis duplication rate of 82.6%(19/23 embryos) was obtained using 1 ng of RNA. CHL polypeptide, with orwithout a FLAG-tag, is active in antagonizing the endogenousventralizing factor (presumably, BMP4) in a similar fashion as CHD.

EXAMPLE 10 Biological Activity of Murine CHL Polypeptide in ES Cells

The ability of CHL polypeptide to inhibit the activity of BMP4 wasassayed in mouse embryonic stem (ES) cell culture as follows. E14 EScells were maintained and differentiated as described by Nakayama etal., 1998, Blood 91: 2283-95, with the exception that 0.9%methylcellulose (Stem Cell Technology) was added to the differentiationmedium. Serum-free differentiation was achieved by replacing the FCSwith Knockout-SR (Gibco BRL), at a concentration of 15%, at thepre-culture stage. The initial cell concentration was between 2500 and4500 cells/ml. Rat SCF was added at 100 ng/ml to all of thedifferentiation cultures. Differentiated cells, aggregated as embryoidbodies, were collected, resuspended in 0.25% collagenase mix (a 1:1mixture of Collagenase D (Boehringer Mannheim) and Collagenase XI(Sigma)) and 15% FCS in PBS, and then incubated for 60 minutes at 37° C.A single cell suspension was then obtained by passing the cells througha 21-gauge needle followed by filtration through a 40-□ mesh. The cellswere spun, resuspended in 0.5% BSA in PBSA at 5×10⁶ cells/ml, and thenstained with 2-20 □g/mL of antibodies against the hematopoietic cellmarkers CD34, Ter119, and CD45 (Pharmingen). The stained samples wereanalyzed on a FACScan (Becton Dickinson). Both Ter119+ erythroid cellformation and CD45+ macrophage cell generation were dependent on theaddition of between 0.5 to 2 ng/ml of human BMP4 protein (R&D Systems).

To assay the activity of the murine CHL-FLAG protein, a BMP4concentration of 0.5 ng/mL was utilized, resulting in approximately ahalf-maximal level of Ter119+ erythroid cell formation during 7 days ofdifferentiation. The CHL-FLAG protein made by the transient transfectionof 293T cells was added at 0.2 to 2 mL/assay, corresponding toapproximately 10 to 100 ng/mL of the CHL-FLAG protein/assay. Decreasedlevels of CD45+ and Ter119+ cells were detected depending upon theamount of CHL-FLAG protein that was added. For example, the percentageof Ter119+ cells was reduced by 35-50% when 50 to 100 ng/mL of CHL-FLAGprotein was added (FIG. 16). This suggests that CHL-FLAG protein isactive, perhaps directly, in inhibiting BMP4 activity.

EXAMPLE 11 Direct Interaction of CHL Polypeptide and BMP4

The direct interaction of Xenopus CHD protein with human BMP4 proteinhas been previously demonstrated (Piccolo et al., 1996, Cell 86:589-98). Similar experiments were performed using murine CHLpolypeptide. An anti-FLAG antibody M2-conjugated agarose gel (Sigma),recognizing the carboxyl-terminus of the CHL-FLAG and CHD-FLAG fusionproteins was used to immunoprecipitate the CHL-BMP4 complex. Bound BMP4protein was quantified by Western blot analysis using an anti-BMP2/4goat polyclonal antibody (Santa Cruz).

Prior to immunoprecipitation, partially purified CHL-FLAG or CHD-FLAGand BMP4 were incubated in TBS (50 mnM Tris HC mnM NaCl, pH 7.4) for 1hour at 4° C. Following incubation of the proteins, anti-FLAG M2affinity gel was added and the reaction incubated for an additional 2hours at 4° C. in an Eppendorf mixer. After which, the agarose gels werespun down for 3 minutes and washed twice in 1 ml PBS. Following additionof gel loading buffer, the reactions were boiled for 10 minutes,electrophoresed through SDS-polyacrylamide gels under reducingconditions, and then electroblotted onto nitrocellulose membranes.

The filter was subsequently blocked with TBST (10 mM Tris-HCl pH7.5,0.9% NaCl, 0.1% Tween-20) containing 3% BSA and incubated in the samebuffer with a 300-fold dilution of the anti-BMP2/4 antibody for 2 hoursat room temperature. The blot was visualized using peroxidase-conjugatedanti-goat secondary antibody (Pierce) and the chemiluminescent ECL kit(Amersham). The co-immunoprecipitation of BMP4 protein with eitherCHL-FLAG or CHD-FLAG suggested that there was a direct physicalinteraction between CHL polypeptide, or CHD, and BMP4 (FIG. 14B).

EXAMPLE 12 BMP4-Dependent Cell Proliferation and Survival

To analyze CHL polypeptide-mediated inhibition of BMP4-dependentproliferation and survival in A5-F stromal cells, 100 □l of test samplediluted in Iscove's modified Dulbecco medium (IMDM), 40 □l of 5×serum-free media containing bovine insulin (500 □g/mi), and 2000 A5-Fmurine bone marrow stromal cells (in a volume of 60 □l in IMDM) wereadded to each well in a 96-well mouse Collagen IV coated plate (BectonDickinson). The culture was then incubated for 72 hours at 37° C. and 5%CO₂. Following incubation, 22 □l of Alamar Blue Cell ProliferationIndicator (Biosource) was added to each well and the culture wasincubated for an additional 24 hours. Following this incubation, thewells were read on a fluorescence microplate reader (BioTech InstrumentsFL500; using the following settings: excitation at 530/25 nm; emissionat 590/35 nm; and sensitivity at 36). In this assay, Alamar Blue CellProliferation Indicator serves as a fluorometric growth indicator ordetector of metabolic activity. Specifically, Alamar Blue CellProliferation Indicator acts as an oxidation-reduction indicator thatfluoresces in response to the chemical reduction of growth mediumresulting from cell growth. In the presence of continued cell growth, areduced environment is maintained in the culture medium, while in thepresence of inhibited cell growth, an oxidized environment ismaintained. Reduction related to cell growth causes the redox indicatorto change from an oxidized form (i.e., non-fluorescent, blue) to areduced form (i.e., fluorescent, red). FIGS. 17-18 illustrate theresults of BMP-4 dependent cell proliferation and survival assaysfollowing incubation of A5-F cells in BMP-4 protein (FIG. 17) or CHLpolypeptide (FIG. 18).

EXAMPLE 13 Expression of CHL Polypeptide in Transgenic Mice

To assess the biological activity of CHL polypeptide, a constructencoding a CHL polypeptide/Fc fusion protein under the control of aliver specific ApoE promoter is prepared. The delivery of this constructis expected to cause pathological changes that are informative as to thefunction of CHL polypeptide. Similarly, a construct containing thefull-length CHL polypeptide under the control of the beta actin promoteris prepared. The delivery of this construct is expected to result inubiquitous expression.

To generate these constructs, PCR is used to amplify template DNAsequences encoding a CHL polypeptide using primers that correspond tothe 5′ and 3′ ends of the desired sequence and which incorporaterestriction enzyme sites to permit insertion of the amplified productinto an expression vector. Following amplification, PCR products are gelpurified, digested with the appropriate restriction enzymes, and ligatedinto an expression vector using standard recombinant DNA techniques. Forexample, amplified CHL polypeptide sequences can be cloned into anexpression vector under the control of the human □-actin promoter asdescribed by Graham et al., 1997, Nature Genetics, 17:272-74 and Ray etal., 1991, Genes Dev. 5:2265-73.

Following ligation, reaction mixtures are used to transform an E. colihost strain by electroporation and transformants are selected for drugresistance. Plasmid DNA from selected colonies is isolated and subjectedto DNA sequencing to confirm the presence of an appropriate insert andabsence of mutation. The CHL polypeptide expression vector is purifiedthrough two rounds of CsCl density gradient centrifugation, cleaved witha suitable restriction enzyme, and the linearized fragment containingthe CHL polypeptide transgene is purified by gel electrophoresis. Thepurified fragment is resuspended in 5 mM Tris, pH 7.4, and 0.2 mM EDTAat a concentration of 2 mg/mL.

Single-cell embryos from BDF1×BDF1 bred mice are injected as described(PCT Pub. No. WO 97/23614). Embryos are cultured overnight in a CO₂incubator and 15-20 two-cell embryos are transferred to the oviducts ofa pseudopregnant CD1 female mice. Offspring obtained from theimplantation of microinjected embryos are screened by PCR amplificationof the integrated transgene in genomic DNA samples as follows. Earpieces are digested in 20 mL ear buffer (20 mM Tris, pH 8.0, 10 mM EDTA,0.5% SDS, and 500 mg/mL proteinase K) at 55° C. overnight. The sample isthen diluted with 200 mL of TE, and 2 mL of the ear sample is used in aPCR reaction using appropriate primers.

At 8 weeks of age, transgenic founder animals and control animals aresacrificed for necropsy and pathological analysis. Portions of spleenare removed and total cellular RNA isolated from the spleens using theTotal RNA Extraction Kit (Qiagen) and transgene expression determined byRT-PCR. RNA recovered from spleens is converted to cDNA using theSuperScript™ Preamplification System (Gibco-BRL) as follows. A suitableprimer, located in the expression vector sequence and 3′ to the CHLpolypeptide transgene, is used to prime cDNA synthesis from thetransgene transcripts. Ten mg of total spleen RNA from transgenicfounders and controls is incubated with 1 MM of primer for 10 minutes at70° C. and placed on ice. The reaction is then supplemented with 10 mMTris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl₂, 10 mM of each dNTP, 0.1 mMDTT, and 200 U of SuperScript II reverse transcriptase. Followingincubation for 50 minutes at 42° C., the reaction is stopped by heatingfor 15 minutes at 72° C. an digested with 2U of RNase H for 20 minutesat 37° C. Samples are then amplified by PCR using primers specific forCHL polypeptide.

EXAMPLE 14 Biological Activity of CHL Polypeptide in Transgenic Mice

Prior to euthanasia, transgenic animals are weighed, anesthetized byisofluorane and blood drawn by cardiac puncture. The samples aresubjected to hematology and serum chemistry analysis. Radiography isperformed after terminal exsanguination. Upon gross dissection, majorvisceral organs are subject to weight analysis.

Following gross dissection, tissues (i.e., liver, spleen, pancreas,stomach, the entire gastrointestinal tract, kidney, reproductive organs,skin and mammary glands, bone, brain, heart, lung, thymus, trachea,esophagus, thyroid, adrenals, urinary bladder, lymph nodes and skeletalmuscle) are removed and fixed in 10% buffered Zn-Formalin forhistological examination. After fixation, the tissues are processed intoparaffin blocks, and 3 mm sections are obtained. All sections arestained with hematoxylin and exosin, and are then subjected tohistological analysis.

The spleen, lymph node, and Peyer's patches of both the transgenic andthe control mice are subjected to immunohistology analysis with B celland T cell specific antibodies as follows. The formalin fixed paraffinembedded sections are deparaffinized and hydrated in deionized water.The sections are quenched with 3% hydrogen peroxide, blocked withProtein Block (Lipshaw, Pittsburgh, Pa.), and incubated in ratmonoclonal anti-mouse B220 and CD3 (Harlan, Indianapolis, Ind.).Antibody binding is detected by biotinylated rabbit anti-ratimmunoglobulins and peroxidase conjugated streptavidin (BioGenex, SanRamon, Calif.) with DAB as a chromagen (BioTek, Santa Barbara, Calif.).Sections are counterstained with hematoxylin.

After necropsy, MLN and sections of spleen and thymus from transgenicanimals and control littermates are removed. Single cell suspensions areprepared by gently grinding the tissues with the flat end of a syringeagainst the bottom of a 100 mm nylon cell strainer (Becton Dickinson,Franklin Lakes, N.J.). Cells are washed twice, counted, andapproximately 1×10⁶ cells from each tissue are then incubated for 10minutes with 0.5 □g CD16/32(FcyIII/II) Fc block in a 20 □L volume.Samples are then stained for 30 minutes at 2-8° C. in a 100 □L volume ofPBS (lacking Ca+ and Mg+), 0.1% bovine serum albumin, an 0.01% sodiumazide with 0.5 □g antibody of FITC or PE-conjugated monoclonalantibodies against CD90.2 (Thy-1.2), CD45R (B220), CD11b(Mac-1), Gr-1,CD4, or CD8 (PharMingen, San Diego, Calif.). Following antibody binding,the cells are washed and then analyzed by flow cytometry on a FACScan(Becton Dickinson).

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the invention as claimed.

1. An isolated polypeptide selected from: (a) a polypeptide comprisingamino acids 1-333 of SEQ ID NO:2; (b) a polypeptide comprising aminoacids 23-333 of SEQ ID NO:2; (c) a polypeptide comprising amino acids1-382 of SEQ ID NO:5; (d) a polypeptide comprising amino acids 23-382 ofSEQ ID NO:5; (e) a polypeptide comprising amino acids 22-452 of SEQ IDNO:8; (f) a polypeptide comprising amino acids 19-452 of SEQ ID NO:8;(g) a polypeptide comprising amino acids 1-452 of SEQ ID NO:8; (h) apolypeptide comprising amino acids 1-447 of SEQ ID NO: 12; (i) apolypeptide that is at least 90% identical to amino acids 23-333 of SEQID NO:2, wherein the identity is over the full length of amino acids23-333 of SEQ ID NO:2, and wherein the polypeptide retains the capacityto bind a bone morphogenetic protein; (j) a polypeptide that is at least90% identical to amino acids 23-382 of SEQ ID NO:5, wherein the identityis over the full length of amino acids 23-382 of SEQ ID NO:5, andwherein the polypeptide retains the capacity to bind a bonemorphogenetic protein; and (k) the polypeptide of any of (a)-(h) whereinthe polypeptide has an amino-terminus truncation, a carboxy-terminustruncation, or both an amino-terminus and a carboxy-terminus truncationwith the exception that amino acids 1-447 of SEQ ID NO: 12 does not havean amino-terminus truncation, wherein the truncated polypeptide retainsthe capacity to bind a bone morphogenetic protein,
 2. An isolatedpolypeptide comprising amino acids amino acids 22-452 of SEQ ID NO:8. 3.An isolated polypeptide comprising amino acids 19-452 of SEQ ID NO:8. 4.An isolated polypeptide comprising amino acids 1-447 of SEQ ID NO: 12.5. The polypeptide of claim 1 that is covalently modified with awater-soluble polymer.
 6. The polypeptide of claim 7, wherein thewater-soluble polymer is selected from the group consisting ofpolyethylene glycol, monomethoxy-polyethylene glycol, dextran,cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol, propyleneglycol homopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols, and polyvinyl alcohol.
 7. A fusionpolypeptide, comprising the polypeptide of claim 1 fused to aheterologous amino acid sequence, wherein the fusion polypeptide retainsthe capacity to bind a bone morphogenetic protein.
 8. The fusionpolypeptide of claim 9, wherein the heterologous amino acid sequence isan Fc domain or fragment thereof.
 9. The fusion polypeptide of claim 10,wherein the Fc domain is an IgG constant domain or fragment thereof. 10.The polypeptide of claim 4 that is covalently modified with awater-soluble polymer.
 11. The polypeptide of claim 4, wherein thewater-soluble polymer is selected from the group consisting ofpolyethylene glycol, monomethoxy-polyethylene glycol, dextran,cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol, propyleneglycol homopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols, and polyvinyl alcohol.
 12. A fusionpolypeptide, comprising the polypeptide of claim 4 fused to aheterologous amino acid sequence, wherein the fusion polypeptide retainsthe capacity to bind a bone morphogenetic protein.
 13. The fusionpolypeptide of claim 14, wherein the heterologous amino acid sequence isan Fc domain or fragment thereof.
 14. The fusion polypeptide of claim14, wherein the Fc domain is an IgG constant domain or fragment thereof.15. The polypeptide of claim 6 that is covalently modified with awater-soluble polymer.
 16. The polypeptide of claim 6, wherein thewater-soluble polymer is selected from the group consisting ofpolyethylene glycol, monomethoxy-polyethylene glycol, dextran,cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol, propyleneglycol homopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols, and polyvinyl alcohol.
 17. A fusionpolypeptide, comprising the polypeptide of claim 6 fused to aheterologous amino acid sequence, wherein the fusion polypeptide retainsthe capacity to bind a bone morphogenetic protein.
 18. The fusionpolypeptide of claim 19, wherein the heterologous amino acid sequence isan Fc domain or fragment thereof.
 19. The fusion polypeptide of claim20, wherein the Fc domain is an IgG constant domain or fragment thereof.20. A composition, comprising the polypeptide of any of claims 1 through21 and a pharmaceutically acceptable formulation agent.
 21. Thecomposition of claim 22, wherein the pharmaceutically acceptableformulation agent is a carrier, adjuvant, solubilizer, stabilizer, oranti-oxidant.